Modulating the production of neurons and/or oligodendrocytes from white matter progenitor cells

ABSTRACT

The present invention relates to a method of modulating production of neurons and/or oligodendrocytes from neural progenitor cells of human white matter and to a method of treating a subject for a condition modulated by underproduction of oligodendrocytes from human white matter. Both of these methods involve administering an agonist or antagonist of one or more molecules set forth in Tables 1 and/or 2 to the neural progenitor cells. Also disclosed is a method of using an inhibitor of sterol synthesis to differentiate oligodendrocyte progenitor cells to oligodendrocytes.

This application is a continuation of U.S. patent application Ser. No.12/368,838, filed Feb. 10, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/985,306, filed Nov. 10, 2004, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/519,310,filed Nov. 10, 2003, each of which is hereby incorporated by referencein its entirety.

This invention was made with government support under grant numbersR01NS33106 and R01NS39559 awarded by NINDS. The government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention is directed to genes differentially expressed byacutely isolated resident progenitor cells of the human white matter.

BACKGROUND OF THE INVENTION

An abundant population of glial progenitor cells resides in the adulthuman subcortical white matter. These cells give rise to myelinogenicoligodendrocytes upon transplantation, yet when removed from the tissueenvironment they behave as multipotential neurogenic progenitors. Toidentify genes that regulate their homeostasis and cell fate decisionsof these adult progenitor cells, the transcriptional profile of A2B5⁺white matter progenitor cells (WMPCs) sorted from human surgicalresections. The profile of each progenitor isolate sorted cellpopulation was then normalized against that of the tissue white matterfrom which it was derived to identify progenitor-enriched transcripts.WMPCs expressed high levels of PDGFaR, GD3 synthase and NG2 prototypicoligodendrocyte progenitor genes, yet they also expressed high levels ofMASH1 and HES1, suggesting a more primitive phenotype. RNAs encoding themembers of several parallel signaling pathways were differentiallyexpressed by WPMCs relative to unsorted cells. These included receptortyrosine phosphate (RTP)-β/ζ, its ligand pleiotrophin, and itsmodulators NrCAM, tenascin R, and the chondroitin sulfate proteoglycans(CSPG2-5); PDGFαR, which induces pleiotrophin; syndecan-3, its membranepartner FGFR3, and its intracellular partner CASK; the BMP inhibitorsneuralin and BAMBI; and the notch intermediates HES1, musashi and FHL1B.When exposed to oxovanadate, an RTP inhibitor, WNPCs ceased expansionand differentiated as oligodendrocytes, validating the central role ofRTP-β/ζ in progenitor self-maintenance. The co-activation of RTP-β/ζ□with these interactive parallel pathways may provide the means by whichadult progenitors are maintained in a multipotential andmitotically-competent state. As such, they may provide targets by whichto perturb cell fate choices by progenitor cells of the adult humanbrain.

A population of nominally glial progenitor cells resides in theparenchyma of the adult human subcortical white matter. These cells maybe defined by A2B5-immunoreactivity, and by their expression offluorescent reporters placed under the control of the CNP2 promoter (Royet al., “Identification, Isolation, and Promoter-defined Separation ofMitotic Oligodendrocyte Progenitor Cells From the Adult HumanSubcortical White Matter.” J Neurosci 19: 9986-95 (1999); Nunes et al.,“Identification and Isolation of Multipotential Neural Progenitor Cellsfrom the Subcortical White Matter of the Adult Human Brain.” Nat Med 9:439-447 (2003)). The cells typically act as oligodendrocyte progenitors,giving rise to myelinogenic oligodendrocytes upon transplantation.However, when removed from the tissue environment, they behave asmultipotential and neurogenic progenitor cells. This observationsuggested that the local tissue environment regulates both theself-renewal and phenotype of parenchymal glial progenitors, such thatthe latter actually represent a pool of multipotential progenitors whosefate is tonically restricted by their local tissue environment. As aresult, the environmental cues presented to these cells, and theirresponsiveness to these signals, may determine not only their mitoticturnover, but also their undifferentiated self-renewal and post-mitoticlineage choices. Yet no studies to date have specifically examined theenvironment of the adult human white matter from the standpoint ofsteady-state cues and cell-specific responsiveness by residentprogenitor cells.

The present invention is directed to overcoming this deficiency in theart.

SUMMARY OF THE INVENTION

The present invention relates to a method of modulating production ofneurons and/or oligodendrocytes from neural progenitor cells of humanwhite matter. This involves administering an agonist or antagonist ofone or more molecules set forth in Tables 1 and/or 2 to the neuralprogenitor cells under conditions effective to modulate production ofneurons and/or oligodendrocytes.

Another aspect of the present invention relates to a method of treatinga subject for a condition modulated by underproduction, dysfunction, orloss of oligodendrocytes from human white matter. This method involvesadministering to the subject an agonist or antagonist of one or moremolecules molecules set forth in Tables 1 and/or 2 under conditionseffective to treat the condition modulated by underproduction,dysfunction, or loss of oligodendrocytes.

Another aspect of the present invention relates to a methoddifferentiating oligodendrocyte progenitor cells to oligodendrocytes.This involves administering an inhibitor of sterol synthesis underconditions effective to differentiate oligodendrocyte progenitor cellsto oligodendrocytes.

To identify genes that regulate both the turnover and fate decisions ofadult glial progenitor cell population in vivo, U95Av2 Affymetrixmicroarrays were used to analyze the transcriptional profile of A2B5⁺white matter progenitor cells (WMPCs), sorted from human white mattersamples derived from surgically-resected adult temporal lobe. Theprofile of each sorted cell population was then normalized against thatof the unsorted dissociate from which it was derived, to identifyWMPC-enriched transcripts that were otherwise under-represented in thewhite matter. By this strategy, several unexpected ligands and receptorsand their attendant signaling pathways were identified that appear touniquely characterize the interaction of oligodendrocyte progenitorcells with the ambient white matter in which they reside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show tyrosine phosphatase inhibition induces oligodendrocytedifferentiation by adult WMPCs. As shown in FIGS. 1A-D, WMPCs weretreated with 0 or 25 ng/ml bpV(phen) for 7 days in vitro; matched wellswere then stained for either A2B5 or O4. FIG. 1E shows the dose responsecurve of the percentage of A2B5⁺ or O4⁺ cells as a function of bpV(phen)dose (±SEM, n=4). Scale bar, 1004.

FIG. 2 shows the signal pathways identified within the adult human WMPC.Over 110 specific genes were significantly and differentially expressedby isolated human WMPCs. The assignment of these genes into coherentsignaling pathways allowed generation of this model, which may predictaspects of the metabolic regulation of WMPCs at steady state, in theadult white matter environment. The signaling pathways predominant inthis model are pleiotrophin signaling via RTPβ/ζ or syndecan-3, notchsignaling, PDGFαR-dependent signaling, and BMP signaling and inhibitionthereof. Genes in color were found to be significantly enriched in whitematter progenitors, compared to unsorted white matter cells.

FIG. 3 shows the role of sterol synethis enzymes and products in thedifferentiation of adult human oligodendrocyte progenitor cells. Theidentification of this gene expression pattern in adult humanoligodendrocyte progenitor cells indicates that inhibition of sterolsynethesis in these cells may lead to oligodendrocyte differentiation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of modulating production ofneurons and/or oligodendrocytes from neural progenitor cells of humanwhite matter. This involves administering an agonist or antagonist ofone or more molecules set forth in Tables 1 and/or 2 to the neuralprogenitor cells under conditions effective to modulate production ofneurons and/or oligodendrocytes.

TABLE 1 Genes Enriched in A2B5-sorted Adult Human WMPCs Compared toUnsorted Dissociate LIGANDS, ANTAGONISTS & SECRETED PROTEINS BMP2 Dpphomologue CHGB chromogranin B (secretogranin 1) BMP7 OP-1 CLU clusterinFRZB SFRP3 MMP16 matrix metalloproteinase 16 (membrane- inserted) NELL1NEL-like 1 (chicken), NRP1 PRSS11 protease, serine, 11 (IGF binding)NELL2 NEL-like 2 (chicken), NRP2 SCG2 secretogranin II (chromogranin C)NRLN1 Neuralin 1 SERPINE2 glia-derived nexin PTN pleiotrophin TIMP4tissue inhibitor of metalloproteinase 4 SLIT1 slit homolog 1(Drosophila) RECEPTORS & DOWNSTREAM COMPONENTS CNR1 cannabinoid receptor1 (brain) ACK1 activated p21cdc42Hs kinase FGFR3 fibroblast growthfactor receptor 3 ADCY8 adenylate cyclase 8 (brain), ADCY3, HBAC1 GABBR1gamma-aminobutyric acid (GABA) B receptor, 1 ARHGEF4 Rho guaninenucleotide exchange factor (GEF) 4 GABRB1 GABA A receptor ARHGEF6Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 GLRB glycinereceptor, beta ARL7 ADP-ribosylation factor-like 7 GPR19 Gprotein-coupled receptor 19 CAP2 adenylyl cyclase-associated protein 2GRIA2 glutamate receptor, ionotropic, AMPA 2 CASKcalcium/calmodulin-dependent serine protein kinase GRIA3 glutamatereceptor, ionotrophic, AMPA 3 DOK5 docking protein 5 GRIK1 glutamatereceptor, ionotropic, kainate 1 INSIG1 insulin induced gene 1 GRIK2glutamate receptor, ionotropic, kainate 2 JIK STE20-like kinase KLRC3killer cell lectin-like receptor MAB21L1 mab-21-like 1 subfamily C,member 3 LDLR low density lipoprotein receptor MAGED1 NRAGE, DLXIN1 LRP1low density lipoprotein-related protein 1 NMA BAMBI(alpha-2-macroglobulin receptor) PDGFRA platelet-derived growth factorreceptor, PKIA protein kinase (cAMP-dependent, catalytic) alphapolypeptide inhibitor alpha TM4SF2 transmembrane 4 superfamily member 2PPAP2B phosphatidic acid phosphatase type 2B TM4SF6 transmembrane 4superfamily member 6 RAB31 RAB31, member RAS oncogene family SHC3neuronal Shc SIAH1 seven in absentia homolog 1 (Drosophila) SPRY2sprouty homolog 2 (Drosophila) CELL ADHESION & EXTRACELLULAR MATRIXMOLECULES ASTN astrotactin PCDH8 protocadherin 8, PAPC, Arcadlin CDH11OB-Cadherin SDC3 syndecan 3 (N-syndecan) CDH13 cadherin 13, H-cadherin(heart) BGN biglycan CDH18 cadherin 18, type 2 COL11A1 collagen, typeXI, alpha 1 CHL1 close homolog of L1CAM COL16A1 collagen, type XVI,alpha 1 CLDN10 claudin 10 CRTL1 cartilage linking protein 1 CLSTN1calsyntenin 1 CSPG2 versican DSCAM Down syndrome cell adhesion moleculeCSPG3 neurocan FLRT2 fibronectin leucine rich transmembrane protein 2CSPG4 NG2 GPM6A glycoprotein M6A CSPG5 neuroglycan C/NGC ITGA7 integrin,alpha 7 PTPRZ1 RPTPzeta/phosphocan KIAA1775 MT-protocadherin SPARCL1SPARC-like 1 (mast9, hevin) NCAM1 NCAM THBS2 thrombospondin 2 NLGN1neuroligin 1 THBS4 thrombospondin 4 NRCAM neuronal cell adhesionmolecule TNR tenascin-R OPCML OBCAM ENZYMES ALDH1A3 aldehydedehydrogenase 1 family, member A3 IDI1 isopentenyl-diphosphate deltaisomerase ALDH5A1 aldehyde dehydrogenase 5 family, member A1 KIAA0455PLASTICITY-RELATED GENE 1/PRG1 (succinate-semialdehyde dehydrogenase)ALDOC Zebrin II/Aldolase C LCK lymphocyte-specific protein tyrosinekinase, p56(lck) B3GNT6 IGAT, IGNT, iGAT, iGNT, BETA3GNTI MOXD1monooxygenase, DBH-like 1 BAAT bile acid Coenzyme A: amino acid N- NME4non-metastatic cells 4, protein expressed in acyltransferase (glycineN-choloyltransferase) CHST10 carbohydrate sulfotransferase 10 PDE8Bphosphodiesterase 8B CKMT1 creatine kinase, mitochondrial 1 (ubiquitous)PFKM phosphofructokinase, muscle CPE carboxypeptidase E PGM1phosphoglucomutase 1 DUSP8 dual specificity phosphatase 8 PRDX2peroxiredoxin 2 ELOVL5 ELOVL family member 5, elongation of long PTPN4protein tyrosine phosphatase, non-receptor chain fatty acids (FEN1/Elo2,SUR4/Elo3-like, type 4 (megakaryocyte) yeast) GAD1 GAD67 SC4MOLsterol-C4-methyl oxidase-like GLDC glycine dehydrogenase (glycinecleavage system SIAT8A GD3 synthase protein P) H105E3 NAD(P) dependentsteroid dehydrogenase-like TRB2 tribbles homolog 2 HMGCR HMG-CoA;3-hydroxy-3-methylglutaryl- Coenzyme A reductase TRANSCRIPTION FACTORS &REGULATORS ASCL1 MASH1 LHX2 LIM homeobox protein 2, LH-2 CROC4transcriptional activator of the c-fos promoter NFIB nuclear factor I/BFHL1 SLIM1 NR2F1 COUP-TFI FOXG1B BF1 NRF NF-kappa B-repressing factorHCFC1 host cell factor C1 SOX13 SRY (sex determining region Y)-box 13HES1 hairy and enhancer of split 1 SOX4 SRY (sex determining regionY)-box 4 HLF hepatic leukemia factor SOX5 SRY (sex determining regionY)-box 5 ING3 inhibitor of growth family, member 3 ZFP36L2 zinc fingerprotein 36, C3H type-like 2 JUN c-JUN OTHER GENES ABCC8 ATP-bindingcassette, sub-family C LPHN3 latrophilin 3 (CFTR/MRP), member 8 ACCN2amiloride-sensitive cation channel 2, neuronal MAP2microtubule-associated protein 2 ACTC actin, alpha, cardiac muscle MEG3maternally expressed 3 AF1Q ALL1-fused gene from chromosome 1q MID1midline 1 (Opitz/BBB syndrome) APOD apolipoprotein D N33 Putativeprostate cancer tumor suppressor ATP1A2 ATPase, Na+/K+ transporting,alpha 2 NCALD neurocalcin delta ATP1B2 ATPase, Na+/K+ transporting, beta2 polypeptide NEBL nebulette ATP2A2 ATPase, Ca++ transporting, cardiacmuscle, NICE-4 NICE-4 protein slow twitch 2 ATP2B4 ATPase, Ca++transporting, plasma membrane 4 NPD009 NPD009 protein BASP1 brainabundant, membrane attached signal NPIP nuclear pore complex interactingprotein protein 1 BC008967 hypothetical gene OIP106 OGT(O-Glc-NActransferase)-interacting protein 106 Kda BSCL2 Bernardinelli-Seipcongenital lipodystrophy 2 OLFM1 olfactomedin 1 (seipin) C11orf8chromosome 11 open reading frame 8 PARD3 par-3 partitioning defective 3homolog CADPS Ca2+-dependent activator protein for secretion PCF11PCF11p homolog CCND1 cyclin D1 (PRAD1: parathyroid adenomatosis 1)PDE4DIP phosphodiesterase 4D interacting protein (myomegalin) COG4component of oligomeric golgi complex 4 PDZK3 PDZ domain containing 3CRMP1 DRP1, DPYSL1, ULIP3 PER1 period homolog 1 (Drosophila) CRY1cryptochrome 1 PER2 period homolog 2 (Drosophila) D2S448 Melanomaassociated gene PM5 pM5 protein DCX doublecortin PNMA2 paraneoplasticantigen MA2 DNAJB1 HSP40 ProSAPiP1 ProSAPiP1 protein DPYSL3 DRP3, CRMP4,ULIP1 RAMP1 receptor (calcitonin) activity modifying protein 1 DZIP1zinc finger DAZ interacting protein 1 RARRES2 retinoic acid receptorresponder (tazarotene induced) 2 EEF1A2 eukaryotic translationelongation factor 1 alpha 2 RBBP6 retinoblastoma binding protein 6 EMU1emilin and multimerin-domain containing protein 1 SCRG1 scrapieresponsive protein 1 EPM2AIP1 EPM2A (laforin) interacting protein 1SEMA5A sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5A EPN2 epsin 2 SEMACAP3 likely ortholog of mouse semaFcytoplasmic domain associated protein 3 F3 coagulation factor III, TFSEZ6L seizure related 6 homolog (mouse)-like FLJ13310 hypotheticalprotein FLJ13310 SLC1A1 solute carrier family 1 (neuronal/epithelialhigh affinity glutamate transporter, system Xag), member 1 GAP43 growthassociated protein 43 SLC1A2 glial high affinity glutamate transporterHIS1 HMBA-inducible, CLP1, HIS1 SMARCD3 SWI/SNF related, matrixassociated, actin dependent regulator of chromatin, subfamily d, member3 HSPH1 HSP105A, HSP105B SRPX sushi-repeat-containing protein, Xchromosome ITM2A integral membrane protein 2A SYT11 synaptotagmin XIKCNB1 potassium voltage-gated channel, Shab-related TARBP1 TAR (HIV) RNAbinding protein 1 subfamily, member 1 KCND3 potassium voltage-gatedchannel, Shal-related THY1 Thy-1 cell surface antigen subfamily, member3 KIAA0062 KIAA0062 protein TNKS tankyrase, TRF1-interactingankyrin-related ADP-ribose polymerase KIAA0354 KIAA0354 gene productTRB@ T cell receptor beta locus KIAA0888 KIAA0888 protein TRIM9tripartite motif-containing 9 KIAA0931 KIAA0931 protein TRO trophinin,magphinin, MAGED3 KIAA0992 Palladin TUBB tubulin, beta polypeptideLAPTM4B lysosomal associated protein transmembrane 4 USP24 ubiquitinspecific protease 24 beta LOC348155 similar to hypothetical proteinLOC283824 YAF2 YY1 associated factor 2 LOH11CR2A loss of heterozygosity,11, chromosomal region 2, gene A Matched profiles of A2B5-sorted WMPCsand the tissue dissociate from a single white matter sample werecompared againsts one another. Significantly enriched genes wereidentified using the resulting expression ratios. Expression of reliablydetected genes, those with at least one present call, were analyzed.Over 250 probes sets were identified that possesed significantlyenriched expression in the WMPCs, i.e. significantly different ratioscompared to unity as determined by a pairwise t-test, p < 0.05, with 20%FDR multiple testing correction. These probe sets were annotated to 210distinct genes, shown here. These genes were annotated using LocusLink,OMIM, and PubMed to assertain possible function in WMPC regulation.

TABLE 2 Genes Depleted from A2B5-sorted Adult Human WMPCs Compared toUnsorted Dissociate LIGANDS, ANTAGONISTS & SECRETED PROTEINS CCL20chemokine (C-C motif) ligand 20 IL1B interleukin 1, beta FGF1 acidic FGFIL1RN interleukin 1 receptor antagonist GRN granulin RECEPTORS &DOWNSTREAM COMPONENTS C3AR1 complement component 3a receptor 1 CCRL2chemokine (C-C motif) receptor-like 2 FCGR2A Fc fragment of IgG, lowaffinity IIa, DOK1 docking protein 1, 62 kDa (downstream of receptor for(CD32) tyrosine kinase 1) IL10RA interleukin 10 receptor, alpha LYNv-yes-1 Yamaguchi sarcoma viral related oncogene homolog LILRB4leukocyte immunoglobulin-like receptor, MPP1 membrane protein,palmitoylated 1, 55 kDa subfamily B (with TM and ITIM domains), member 4CCR1 chemokine (C-C motif) receptor 1 SOCS4 suppressor of cytokinesignaling 4 CCR5 chemokine (C-C motif) receptor 5 ENZYMES BLVRBbiliverdin reductase B (flavin reductase LIPA lipase A, lysosomal acid,cholesterol esterase (NADPH)) (Wolman disease) GPX1 glutathioneperoxidase 1 MEP1A meprin A, alpha (PABA peptide hydrolase) GSTO1glutathione S-transferase omega 1 PTP4A2 protein tyrosine phosphatasetype IVA, member 2 KYNU kynureninase (L-kynurenine hydrolase)TRANSCRIPTION FACTORS & REGULATORS HIF1A hypoxia-inducible factor 1,alpha PPARG PPAR gamma subunit (basic helix-loop-helix transcriptionfactor) TFEC transcription factor EC OTHER GENES CLIC1 chlorideintracellular channel 1 HLA-DPA1 major histocompatibility complex, classII, DP alpha 1 FER1L3 fer-1-like 3, myoferlin (C. elegans) HLA-DQB1major histocompatibility complex, class II, DQ beta 1 KIAA0053 KIAA0053gene product HLA-DRB1 major histocompatibility complex, class II, DRbeta 1 LOC253982 hypothetical protein LOC253982 KIF1C kinesin familymember 1C LPXN leupaxin LCP1 lymphocyte cytosolic protein 1 (L-plastin)LY86 lymphocyte antigen 86 LCP2 lymphocyte cytosolic protein 2 (SH2domain containing leukocyte protein of 76 kDa) TRIM44 tripartitemotif-containing 44 LGALS1 lectin, galactoside-binding, soluble, 1(galectin 1) APOC2 apolipoprotein C-II PXR1 peroxisome receptor 1 BCL2L2BCL2-like 2 RNASE6 ribonuclease, RNase A family, k6 FABP4 fatty acidbinding protein 4, adipocyte S100A11 S100 calcium binding protein A11(calgizzarin) GAS7 growth arrest-specific 7 TRIM38 tripartitemotif-containing 38 HBA1 hemoglobin, alpha 1 UCP2 uncoupling protein 2(mitochondrial, proton carrier) HBG1 hemoglobin, gamma A Matchedprofiles of A2B5-sorted WMPCs and the tissue dissociate from a singlewhite matter sample were compared against one another. Significantlydepleted genes were identified using the resulting expression ratios.Expression of reliably detected genes, those with at least one presentcall, were analyzed. 51 probes sets were identified that possessedsignificantly lower expression in the WMPCs than the tissue dissociate,i.e. significantly different ratios compared to unity as determined by apairwise t-test, p < 0.05, with 20% FDR multiple testing correction.These probe sets were annotated to 51 distinct genes, shown here. Thesegenes were annotated using LocusLink, OMIM, and PubMed to assertainpossible function in WMPC regulation.

Agonists and antagonists in accordance with the present invention arewell known to those skilled in the art.

Examples of gamma-secretase inhibitors include: L-685,458 (Shearman, et.al., “L-685,458, an Aspartyl Protease Transition State Mimic, is aPotent Inhibitor of Amyloid β-Protein Precursor γ-Secretase Activity,”Biochem. 39:8698-704 (2000); Doerfler, et. al., “Presenilin-Dependentγ-Secretase Activity Modulates Thymocyte Development,” Proc. Nat'l Acad.Sci USA 98(16): 9312-17 (2001), which are hereby incorporated byreference in their entirety); MG132 (Klafki, et. al., “The CarboxylTermini of β-Amyloid Peptides 1-40 and 1-42 are Genereated by Distinctγ-Secretase Activities,” J. Biol. Chem. 271(45): 28655-59 (1996);Strooper, et. al., “A Presenilin-1-Dependent γ-Secretase-Like ProteaseMediates Release of Notch Intracellular Domain,” Nature 398:518-22(1998), which are hereby incorporated by reference in their entirety),Compounds A-G in Seiffert, et. al., “Presenilin-1 and -2 are MolecularTargets for γ-Secretase Inhibitors,” J. Biol. Chem. 275(44): 34086-91(2000), which is hereby incorporated by reference in its entirety;compounds-2 and-3 in Doerfler, et. al., “Presenilin-Dependentγ-Secretase Activity Modulates Thymocyte Development,” Proc. Nat'l Acad.Sci USA 98(16): 9312-17 (2001), which is hereby incorporated byreference; MD28170 (Citron, et. al., “Evidence that the 42- and 40-AminoAcid Forms of Amyloid β Brotein are Generated from the β-AmyloidPrecursor Protein by Different Protease Activities,” Proc. Nat'l Acad.Sci. USA 93: 13170-75 (1996); De Strooper, et. al., “APresenilin-1-Dependent γ-Secretase-Like Protease Mediates Release ofNotch Intracellular Domain,” Nature 398:518-22 (1998), which are herebyincorporated by reference in their entirety); difluoro ketone compoundCM115 (Wolfe, et. al., “Peptidomimetic Probes and Molecular ModelingSuggest that Alzheimer's γ-Secretase is an Intramembrane-CleavingAspartyl Protease,” Biochem. 38:4720-27 (1999), which is herebyincorporated by reference in its entirety); MW167 (De Strooper, et. al.,“A Presenilin-1-Dependent γ-Secretase-Like Protease Mediates Release ofNotch Intracellular Domain,” Nature 398:518-22 (1998); Wolfe, et. al.,“Peptidomimetic Probes and Molecular Modeling Suggest that Alzheimer'sγ-Secretase is an Intramembrane-Cleaving Aspartyl Protease,” Biochem.38:4720-27 (1999), which are hereby incorporated by reference in theirentirety); CM115 (Wolfe, et. al., “Peptidomimetic Probes and MolecularModeling Suggest that Alzheimer's γ-Secretase is anIntramembrane-Cleaving Aspartyl Protease,” Biochem. 38:4720-27 (1999);Hadland, et. al., “γ-Secretase Inhibitors Repress ThymocyteDevelopment,” Proc. Nat'l Acad. Sci. USA 98(13): 7487-91 (2001)(compound11)), which are hereby incorporated by reference in their entirety);DAPT or N—[N-(3,5-difluoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butylester (Dovey, et. al., “Functional Gamma-Secretase Inhibitors ReduceBeta-Amyloid Peptide Levels in Brain,” J. Neurochem. 76: 173-81 (2001);Geling, et. al., “A γ-Secretase Inhibitor Blocks Notch Signaling in vivoand Causes a Severe Neurogenic Phenotype in Zebrafish,” EMBO Reports3(7): 688-94 (2002), which are hereby incorporated by reference in theirentirety); and various γ-secretase inhibitors in the Calbiochem Catalogas follows:

Cat. No. Product Name 101500 AEBSF, Hydrochloride 171601 APP β-SecretaseInhibitor 196000 Bafilomycin A1, Streptomyces griseus 496000 OM99-2516485 Pepstatin A Methyl Ester 565777 γ-Secretase Inhibitor XVI 565749β-Secretase Inhibitor II 565780 β-Secretase Inhibitor III 565750γ-Secretase Inhibitor I 565755 γ-Secretase Inhibitor II 565760γ-Secretase Inhibitor III 565761 γ-Secretase Inhibitor IV 565762γ-Secretase Inhibitor V 565763 γ-Secretase Inhibitor VI 565770γ-Secretase Inhibitor IX (DAPT, see above) 565771 γ-Secretase InhibitorX (L-685, 458, see above) 565772 γ-Secretase Inhibitor XI 565773γ-Secretase Inhibitor XII 565774 γ-Secretase Inhibitor XIII 565775γ-Secretase Inhibitor XIV 565778 γ-Secretase Inhibitor XVII 565779γ-Secretase Inhibitor XVIII 565765 γ₄₀-Secretase Inhibitor I 565766γ₄₀-Secretase Inhibitor II 565787 γ-Secretase Inhibitor XIX

Examples of FGFR3 inhibitors include: PD1703074 (Bansal, et. al.,“Specific Inhibitor of FGF Receptor Signaling: FGF-2-Mediated Effects onProliferation, Differentiation, and MAPK Activation are Inhibited byPD173074 in Oligodendrocyte-Lineage Cells,” J. Neurosci. Res. 74: 486-93(2003) and Hamby, et. al., “Structure-Activity Relationships for a NovelSeries of Pyrido[2,3-d]pyrimidine Tyrosine Kinase Inhibitors,” J. Med.Chem. 40:2296-303 (1997)(compound 4e), which are hereby incorporated byreference in their entirety) and SU5402 (Mohammadi, et. al., “Structuresof the Tyrosine Kinase Domain of Fibroblast Growth Factor Receptor inComplex with Inhibitors,” Science 276: 955-60 (1997) and Mueller, et.al., “Fibroblast Growth Factor Signaling Regulates Pillar CellDevelopment in the Organ of Corti,” J. Neurosci. 22(21): 9368-77 (2002),which are hereby incorporated by reference in their entirety).

Suitable bone morphogenic protein antagonists include: AMN (amnionlesshomolog); BAMB1 (NMA); BMP1 (TLD); CER1 (Cerebrus); CHRD (chordin);CHRDL1 (Neutralin-1); CHRDL1 (Chordin-like 2); CRIM1 (cystein-rich motorneuron-1); FLJ38607 Dante/Coco homolog; FST (follistatin); FLTL1(follistatin-like 1); FLTL3 (follistatin-like 3); FLTL4(follistatin-like 4); FLTL5 (follistatin-like 5); GREM1 (gremlin); GREM2(PRDC orthologue); IGFBP7 (follistatin-like 2/MAC25); LOC286015 (likeKielin); NBLI (DAN); NOG (noggin); SOST (sclerostin); TLL1 (tolloid-like1); TLL2 (tolloid-like 2); TMEFF1 (transmembrane protein with EGF-likeand two follistatin-like domains); TMEFF2 (transmembrane protein withEGF-like and two follistatin-like domains 2); and TWSG1 (twistedgastrulation).

Suitable platelet-derived growth factor receptor (PDGFR) inhibitorsinclude STI571 or CGP 57148B(4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)o-2-yrimidinyl]amino]-pheny]benzamidemethanesulfonate) (Kilic, et. al., “Intracranial Inhibition ofPlatelet-derived Growth Factor-Mediated Glioblastoma Cell Growth by anOrally Active Kinase Inhibitor of the 2-Phenylaminopyrimidine Class,”Cancer Res. 60: 5143-50 (2000) and Uhrboom, et. al., “Dependence ofAutocrine Growth Factor Stimulation in Platelet-Derived GrowthFactor-B-Induced Mouse Brain Tumor Cells,” Int. J. Cancer 85: 398-406(2000), which are hereby incorporated by reference in their entirety)and the following compounds from the Calbiochem Catalog, which is herebyincorporated by reference in its entirety:

Cat. No. Product Name 521230 PDGF Receptor Tyrosine Kinase Inhibitor I521231 PDGF Receptor Tyrosine Kinase Inhibitor II 521232 PDGF ReceptorTyrosine Kinase Inhibitor III

RTP-zeta (also referred to herein as RTP-β or RTP β/ζ) inhibitorsinclude the following compounds from Calbiochem Catalog, which is herebyincorporated by reference in its entirety:

Cat. No. Product Name 203701 bpV(HOpic) 203695 bpV(phen) 203705 bpV(pic)217691 CDC25 Phosphatase Inhibitor BN82002 322130 DMHV 263200Dephostatin 263202 3,4-Dephostatin 263203 3,4-Dephostatin, Ethyl 521000Phenylarsine Oxide 540215 Protein Tyrosine Phosphatase CD45 Inhibitor540200 Protein Tyrosine Phosphatase Inhibitor I 540205 Protein TyrosinePhosphatase Inhibitor II 540210 Protein Tyrosine Phosphatase InhibitorIII 540211 Protein Tyrosine Phosphatase Inhibitor IV 557322 RK-682,Streptomyces sp. 567565 Sodium Stibogluconate 203694 bpV(bipy)

These molecules can modulate oligodendrocyte progenitor mobilization,division, proliferation, differentiation, and/or self-maintenance. Inaddition, they can modulate oligodendrocyte maturation, differentiation,myelin production, and/or axonal myelination.

Preferably, the neural progenitor cells are oligodendrocyte progenitorcells. These cells can be derived from a post-natal human, fetal, or anadult human.

Administration can be carried out in vivo or in vitro.

Another aspect of the present invention relates to a method of treatinga subject for a condition modulated by underproduction, dysfunction, orloss of oligodendrocytes from post-natal or adult human white matter.This method involves administering to the subject an agonist orantagonist of one or more molecules molecules set forth in Tables 1and/or 2 under conditions effective to treat the condition modulated byunderproduction, dysfunction, or loss of oligodendrocytes.

This embodiment can be carried out with the same agonists andantagonists of the same molecules described above.

Conditions modulated by underproduction, dysfunction, or loss ofoligodendrocytes from post-natal or adult human white matter include thepediatric leukodystrophies, the lysomal storage diseases, congenitaldysmyelination, cerebral palsy, inflammatory demyelination (e.g.,multiple sclerosis), post-infectious and post-vaccinialleukoencephalitis, radiation- or chemotherapy-induced white matterdamage, and vascular demyelination (e.g., stroke, trauma, hypertensiveand diabetic leukoencephalopathy, spinal cord stroke and trauma, andspinal cord compression).

The compounds of the present invention can be administered orally,parenterally, for example, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, or byapplication to mucous membranes, such as, that of the nose, throat, andbronchial tubes. They may be administered alone or with suitablepharmaceutical carriers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they may be enclosed in hard or soft shell capsules,or they may be compressed into tablets, or they may be incorporateddirectly with the food of the diet. For oral therapeutic administration,these active compounds may be incorporated with excipients and used inthe form of tablets, capsules, elixirs, suspensions, syrups, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compound in these compositionsmay, of course, be varied and may conveniently be between about 2% toabout 60% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions according to the presentinvention are prepared so that an oral dosage unit contains betweenabout 1 and 250 mg of active compound.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

These active compounds may also be administered parenterally. Solutionsor suspensions of these active compounds can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols such as, propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds of the present invention may also be administered directlyto the airways in the form of an aerosol. For use as aerosols, thecompounds of the present invention in solution or suspension may bepackaged in a pressurized aerosol container together with suitablepropellants, for example, hydrocarbon propellants like propane, butane,or isobutane with conventional adjuvants. The materials of the presentinvention also may be administered in a non-pressurized form such as ina nebulizer or atomizer.

Another aspect of the present invention relates to a methoddifferentiating oligodendrocyte progenitor cells to oligodendrocytes.This involves administering an inhibitor of sterol synthesis underconditions effective to differentiate oligodendrocyte progenitor cellsto oligodendrocytes. Examples of suitable inhibitors of sterol synthesisinclude lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin,cerivastatin, and rosuvastatin. The compounds can be formulated andadministered in substantially the manner described above. See also FIG.3.

EXAMPLES Example 1 Adult Human Subcortical White Matter

Adult human subcortical white matter was obtained from temporal lobetissue removed from 48 patients at craniotomy, principally formedication-refractory epilepsy (age 17-56 years; 5 males and 3 female).Samples were obtained from patients who consented to tissue use underprotocols approved by the New York Hospital-Cornell, ColumbiaPresbyterian Hospital, and University of Rochester-Strong MemorialHospital Institutional Review Boards. The tissues were prepared andwhite matter progenitor cells freshly isolated as previously described(Nunes et al., “Identification and Isolation of Multipotential NeuralProgenitor Cells from the Subcortical White Matter of the Adult HumanBrain.” Nat Med 9: 439-447 (2003), which is hereby incorporated byreference in its entirety). Briefly, samples were minced into PIPESsolution (in mM: 120 NaCl, 5 KCl, 25 glucose, and 20 PIPES), thendigested in papain-PIPES (11.4 U/ml papain; Worthington, Freehold, N.J.)and DNase I (10 U/ml; Sigma, St. Louis, Mo.), on a shaker for 1.5 hr at37° C. The cells were collected by centrifugation at 200×g in an IECCentra-4B centrifuge, resuspended in DMEM/F-12/N1 with DNase I (10U/ml), and incubated for 30 min at 37° C. The samples were again spun,and their pellets recovered in 2 ml of DMEM/F-12/N1. They were thendissociated by sequentially triturating for 20, 10, and 5 times,respectively, through three glass Pasteur pipettes fire polished todecreasing bore diameters. The cells were passed through a 40 μm meshinto DMEM/F-12/N1, with 10% plasma-derived fetal bovine serum (PD-FBS;Cocalico Biologicals, Reamstown, Pa.) to stop the enzymaticdissociation. The cells were then suspended in DMEM/F12/N1 and incubatedin A2B5-antibody containing supernatant (clone 105; American TypeCulture Collection, Manassas, Va.) for 30-45 min at 4° C. on a shaker.The cells were washed 3× with PBS containing 0.5% bovine serum albuminand 2 mM EDTA, then incubated with 1:4 diluted microbead-tagged ratanti-mouse IgM antibody (MACS, Miltenyi Biotech) for 30 min at 4° C. ona shaker. The A2B5⁺ cells were washed, resuspended, and separated usingpositive selection columns, type MS+/RS+ or LS+/VS+ (MACS, MiltenyiBiotech). The total number of viable cells was determined using calcein(Molecular Probes).

Example 2 Affymetrix GeneChip Protocol

Immediately after sorting, RNA was extracted with Trizol (Invitrogen)and then purified using RNeasy (Qiagen), both according tomanufacturer's specifications. 100 ng of total RNA was amplified usingAffymatrix's small sample protocol (GeneChip® Eukaryotic Small SampleTarget Labeling Technical Note), and 15 μg of cRNA was used on eachU95Av2 GeneChip.

Example 3 Analysis of GeneChip Expression Data

Image files were processed using MAS5.0 to produce CHP files. Imageswere masked to remove streaks or smears present, and no scaling of datawas performed during analysis. Data was then imported into GeneSpring(5.0, Silicon Genetics) and per chip normalization performed (using the50th percentile of all measurements in that sample). Calculation of geneexpression ratios was then performed by comparing the expression patternof each A2B5-sorted sample to that of the unsorted population from whichit had been extracted. This comparison effectively normalizedsample-to-sample variation. The arithmetic mean ratio of A2B5-sorted tounsorted was then calculated from three separate patients. An estimateof error was generated using the Rocke-Lorenzato global error model,which takes into account the variability in the expression level ofindividual genes, compared to that of the entire data set. As a result,lower and more variably expressed genes are given larger error values,and are thus less likely to be deemed significant using statisticalcriteria.

Example 4 Statistical Assignment of Differential Expression

Transcripts deemed significantly enriched or depleted in the sorted cellpool fulfilled the criterion that their sorted: unsorted expressionratios differed significantly from 1; this was effectively a pairedt-test of expression ratios. A Benjamini and Hochberg False DiscoveryRate (FDR) of 20% was selected empirically; at that level, it wasvalidated that 15 of 18 nominally-enriched genes subjected to qPCRvalidation were indeed enriched, while the other 3 were undetectable inthe RNA obtained from unsorted cells, thus precluding ratiodetermination.

Example 5 Annotation of Probe Sets

Qualifying probe sets for each gene on the Affymetrix Human U95Av2 chipwere identified using annotations available from NetAffx and Ensembl.Probe sets with conflicting annotations were verified by BLAST analysisof probe target sequence to the human genome. This process excludedmis-annotated probe sets. Annotation and further data analysis was thenperformed within an in-house Microsoft Access database.

Example 6 Real-Time PCR

The chosen genes validated by quantitative RT-PCR were designed toefficiently test the model generated based on array data alone (See FIG.1). Primers and probes were either designed using Primer express(Applied Biosystems) or obtained as Assays-on-Demand directly fromApplied Biosystems. For each sample, four separate reverse transcriptionreactions of 25 ng total RNA were performed as per manufacturer'sprotocol and the resulting cDNA diluted to 100 pg/μl. Four separatereal-time PCR reactions with 500 pg/reaction, in addition 2 no-RTcontrol reactions were performed to check for RNA-independent productamplification. For taqman real-time PCR, a 900 nM concentration offorward and reverse primers, and 250 nM FAM-labeled MGB probes. For SYBRGreen real-time PCR, 300 nM forward and reverse primers were used. Human18S RNA was used as an endogenous control, as described by themanufacturer (ABI). The relative abundance of transcript expression wascalculated following normalization of the C_(t) value to the matchedunsorted white matter dissociate control, and the final expression ratiothen normalized to the endogenous control. The mean, standard error, andsignificance testing of the individual samples were calculated by firstperforming a log transformation on the ratio data. The values presentedin the tables are the anti-log of these values. Significance was testedusing two-way one-sample t-test against a null ratio of 1 (n=4).

Example 7 Tyrosine Phosphatase Inhibition

WMPCs were distributed onto 12-well plates coated with poly-L-ornithineand fibronectin at 5×10⁴ cells/ml in DMEM/F12/N1 supplemented with 10ng/ml bFGF (Sigma), 10 ng/ml PDGF-AA (Sigma), and 2 ng/ml NT3 (R&DSystems). Stock solutions of 1 μM bpV(phen) (potassiumbisperoxo(1,10-phenanthroline)oxovanadate (V); Calbiochem) were preparedbefore each use. Cells were exposed to concentrations of 0, 1, 5, 10,and 25 ng/ml of bV(phen) immediately upon plating, and every 2 daysthereafter for 7 day in vitro.

Example 8 Immunocytochemistry

Cultures were exposed continuously to 10 μg/ml BrdU beginning 24 hoursbefore fixation. After 7 days in vitro, A2B5 and O4 were immunolabeledas previously described (Roy et al., “Identification, Isolation, andPromoter-defined Separation of Mitotic Oligodendrocyte Progenitor Cellsfrom the Adult Human Subcortical White Matter,” J. Neurosci 19:9986-95(1999), which is hereby incorporated by reference in its entirety). Formultiple antigen labeling, O4 and A2B5 were localized on live cells thatwere then fixed with 4% paraformaldehyde and immunostained for BrdU. O4supernatant was used at a dilution of 1:100 and monoclonal antibody A2B5supernatant (clone 105, American Type Culture Collection) was used in a1:1 ratio with DMEM/F12/N, both for 40 minutes at 4° C. Rat anti-BrdUantibody (Harlan) was used at a dilution of 1:200. Fixed cultures werecounterstained with DAPI (10 ng/ml; Molecular Probes). The number ofA2B5 and O4 stained and unstained cells were counted in 10 randomlychosen fields at each dosage level, from individual replicate samples(n=4). Statistical significance was assessed by one-way repeatedmeasures analysis of variance (ANOVA), followed by Tukey's multiplecomparisons test (GraphPad Prism 3.0, p<0.05).

Example 9 Adult Human WMPCs Expressed Oligodendrocyte Progenitor MarkerGenes

Adult human subcortical white matter progenitor cells (WMPCs) wereenriched by magnetic-activated cell sorting (MACS) using the A2B5 markeras previously described (Roy et al., “Identification, Isolation, andPromoter-defined Separation of Mitotic Oligodendrocyte Progenitor CellsFrom the Adult Human Subcortical White Matter.” J Neurosci 19: 9986-95(1999); Nunes et al., “Identification and Isolation of MultipotentialNeural Progenitor Cells from the Subcortical White Matter of the AdultHuman Brain.” Nat Med 9: 439-447 (2003), which are hereby incorporatedby reference in their entirety). From four epileptic temporal loberesections cases, between 5×10⁵ and 1×10⁶ A2B5⁺ cells, that comprisedroughly 3% of all viably dissociated white matter cells, were obtained.To identify those genes whose expression distinguishes the A2B5⁺ WMPCpopulation from the other glial subtypes present in normal human adultwhite matter, microarray analysis was performed on both RNA extractedfrom WMPCs immediately after sorting and RNA extracted from the specificunsorted dissociates from which the sorts were derived. Beginning withat least 100 ng of total RNA per isolate, two rounds of RNAamplification were performed prior to hybridization to AffymetrixHG-U95Av2 GeneChips using Affymetrix's small sample protocol. Followingmicroarray-wide normalization, the expression of individual genes ineach WMPC isolate was normalized against that of the unsorted whitematter dissociate from which it was derived, and the mean expressionratio calculated from the individual samples.

To analyze the microarray data, the expression of several known markergenes differentially expressed by glial progenitor cells was firstdetermined (Table 3).

TABLE 3 Marker Gene Expression Profile of A2B5 + WMPCs Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3)Oligodendrocyte Progenitor CSPG4 (NG2) 38004 _(—) at 19.41 ± 2.62 PDGFRA 1731 _(—) at ¹ 11.18 ± 0.89  SIAT8A (GD3 synthase) 40678 _(—) at5.25 ± 0.89 Oligodendrocyte Lineage CNP (CNPase) 612_s_at 1.64 ± 0.21NKX2-2 33605_at 1.53 ± 0.42 OLIG2 40624_at 1.71 ± 0.39 PLP1 (PLP/DM20)41158_at 1.21 ± 0.19 QKI 39759_at 1.06 ± 0.29 SOX10 36018_at 1.19 ± 0.26Myelinating Oligodendrocyte GALC 33936_at 1.07 ± 0.20 MAG 38558_at 1.22± 0.24 MAL 38051_at 0.81 ± 0.14 MBP 35817_at 1.32 ± 0.23 MOBP 38499_s_at0.28 ± 0.15 MOG 37868_s_at 0.52 ± 0.25 Astrocyte AQP4 40793_s_at 1.37 ±0.98 AQP9 34435_at 0.29 ± 0.19 GFAP 40185_at 1.70 ± 0.33 GLUL (glutaminesynthase) 40522_at 0.86 ± 0.15 S100B 235_at 1.00 ± 0.14 TNC (Tenascin C)32818_at 1.40 ± 1.14 Neuronal progenitor/stem cell ² ASCL1 (MASH1) 40544_(—) g _(—) at 12.32 ± 1.72  DCX (doublecortin) 34382 _(—) at 7.56 ±2.91 HES1 37393 _(—) at 5.13 ± 1.27 Neural lineage ² ELAVL3 (HuC)38512_r_at 1.13 ± 0.19 ELAVL4 (HuD) 40380_at 2.50 ± 0.54 MAP2 35422_at0.92 ± 0.49 1972 _(—) s _(—) at 4.17 ± 0.56 NEF3 (neurofilament 32512_at1.33 ± 0.13 medium) TUBA3 (Tα1 tubulin) 40567_at 0.84 ± .016 Endothelial² CDH5 (VE-Cadherin) 37196_at 0.58 ± 0.20 TEK (TIE2) 1595_at 0.82 ± 0.56Microglial CD68 33390_at 0.49 ± 0.12 CD86 36270_at 0.27 ± 0.40 HLA-DRA37039_at 0.22 ± 0.14 HLA-DRB1 41723_s_at 0.22 ± 0.12 Genes/probe sets inbold indicate significant enrichment in WMPCs over unsorted dissociatedwhite matter cells. ¹ For genes with multiple Affymetrix probe sets, theprobe set with the most significant ratio of expression is shown. ²SOX1, DLX2/5, NEFL (neurofilament light) and VWF (von Willebrandsfactor) are not detected in either A2B5+ WMPCs or unsorted WM cells butare detected in human fetal VZ tissue.The marker used to isolate adult WMPCs, the monoclonal A2B5 (Eisenbarthet al., “Monoclonal Antibody to a Plasma Membrane Antigen of Neurons,”Proc Natl Acad Sci USA 76:4913-4917 (1979) and Roy et al.,“Identification, Isolation, and Promoter-defined Separation of MitoticOligodendrocyte Progenitor Cells from the Adult Human Subcortical WhiteMatter,” J Neurosci 19:9986-95 (1999), which are hereby incorporated byreference in their entirety) recognizes G_(Q) and G_(T3) gangliosidesand their O-acetylated derivatives (Farrer et al., “GT3 and itsO-Acetylated Derivative are the Principal A2B5-Reactive Gangliosides inCultured O2A Lineage Cells and are Down-Regulated Along with O-AcetylGD3 During Differentiation to Oligodendrocytes.” J Neurosci Res 57:371-380 (1999), which is hereby incorporated by reference in itsentirety). It was found that the expression of GD3 synthase (SIAT8A),the enzyme that catalyzes the transfer of sialic acid from CMP-sialicacid to GM3 and by which GD3 and GT3 are generated was significantlyenriched in the WMPC pool. This observation was confirmed with real-timeRT-PCR analysis (qPCR) of GD3 synthase mRNA levels followingnormalization to 18S ribosomal RNA (one sample t-test, H₁₀=1, p<0.01;Table 4).

TABLE 4 Real Time RT-PCR Validation of Significantly Enriched MarkerGene Ratio of mRNA expression A2B5+ WMPCs:unsorted WM qPCR (n = 3-4)Oligodendrocyte Progenitor CSPG4 (NG2) 15.05 (13.66-16.57; p < 0.001) ¹PDGFRA 22.67 (19.30-26.61; p < 0.001) SIAT8A (GD3 synthase) 9.39(8.62-10.23; p < 0.01) Neuronal progenitor/stem cell ASCL1 (MASH1) 18.67(15.97-21.82; p < 0.05) HES1 12.52 (11.86-13.21; p < 0.001) ¹ Ranges inparenthesis indicates plus/minus one standard deviation.Furthermore, microarray analysis revealed strong expression of PDGFαRand NG2 (CSPG4), two canonical markers of oligodendrocyte progenitors invivo which were confirmed by qPCR (Tables 3 and 4).

The oligodendrocyte progenitor lineage bHLH transcription factors olig2and Nkx2.2 were also detected in the WMPC profile. However, neither genewas significantly enriched compared to the unsorted white matterpresumably since mature oligodendrocytes also express olig2 and Nkx2.2(Lu et al., “Sonic Hedgehog—Regulated Oligodendrocyte Lineage GenesEncoding bHLH Proteins in the Mammalian Central Nervous System,” Neuron25: 317-29 (2000); Watanabe et al., “Transient Upregulation of Nkx2.2Expression in Oligodendrocyte Lineage Cells During Remyelination,” Glia46: 311-322 (2004), which are hereby incorporated by reference in theirentirety). Similarly, more mature oligodendrocytic transcripts,including CNP and the myelin protein genes, myelin basic protein (MBP)and proteolipid protein (PLP1), were under-expressed by WMPCs relativeto their parental white matter. Markers of other white matterphenotypes, namely astrocytes, microglia, and endothelial cells, wereeither unenriched or relatively depleted in WMPCs (Table 3). Thus, thetranscriptional profile of A2B5-sorted WMPCs exhibited the differentialexpression of a number of genes previously associated witholigodendrocyte progenitor cells.

Interestingly, several markers of early neural cell growth and migrationwere noted to be differentially expressed by WMPCs. Doublecortin (DCX),which is expressed on migrating immature cells during development,was >8-fold enriched in WMPCs. GAP43, a growth andregeneration-associated marker of process extension, was significantlyenriched >4-fold in WMPCs, confirming earlier reports of GAP43'sexpression by rodent oligodendrocyte progenitors (Curtis et al.,“Down-regulation of GAP-43 During Oligodendrocyte Development and Lackof Expression by Astrocytes In Vivo: Implications for MacroglialDifferentiation,” Eur J Neurosci 3:876-886 (1991; Fanarraga et al., O-2AProgenitors of the Mouse Optic Nerve Exhibit a Developmental Pattern ofAntigen Expression Different from the Rat,” Glia 15:95-104 (1995), whichare hereby incorporated by reference in their entirety). GAD67 mRNA,which encodes glutamate decarboxylase (GAD) and, as such serves as amarker of GABA production, was enriched >8 fold in A2B5-sorted WMPCs.Although GABA expression has previously not been described inoligodendrocyte lineage cells, GAD expression by these cells may havereflected their potential to generate GABAergic neurons when cultured inlow density (Nunes et al., “Identification and Isolation ofMultipotential Neural Progenitor Cells from the Subcortical White Matterof the Adult Human Brain,” Nat Med 9:439-447 (2003), which is herebyincorporated by reference in its entirety).

Example 10 Adult WMPCs are Transcriptionally Distinct From the LocalWhite Matter Environment

The Affymetrix U95Av2 GeneChip analyzes the expression of approximately8,500 genes. 53% and 56% of the represented genes were present in atleast one sample of the A2B5-sorted WMPC and unsorted dissociatetranscript pools, respectively. The degree of overlap was large; 92% ofthose genes expressed in the A2B5-sorted pool were detected in theunsorted dissociate. A set of genes whose expression was significantlyenriched in the A2B5-sorted WMPC-enriched population compared to theunsorted white matter dissociate was next identified. Using Genespring(Silicon Genetics) to analyze to array data base, those probe sets thatwere deemed ‘absent’ in all three A2B5-sorted profiles were removed. Theremainder comprising reproducibly hybridized oligonucleotides were usedto generate a list of probe sets whose expression was significantlyhigher in sorted cells than unsorted dissociate. The resulting list ofapproximately 250 probe sets (<5% of total) was then pruned by removingthose that were either ambiguously annotated as mapping to multiplegenes or were novel (by virtue of not yet having been annotated to NCBILocusLink identifiers). The remaining probe sets were annotated to 210distinct genes (Table 1). For each identified gene, additional probesets were then identified. Transcripts depleted from the A2B5-sortedWMPC-enriched population were determined by the same analysis procedureby inverting the expression ratios in the A2B5-sorted pool. The numberof depleted transcripts was much smaller with only 51 probe setsidentified that mapped to 51 distinct genes (Table 2).

The frequency of functionally related transcripts was next examined todetermine relevant functional categories of genes. Over representedfunctional categories in the A2B5-sorted WMPC cell profile weredetermined by comparison with the entire population of genes on theHG-U95Av2 microarray. Using the EASE software tool (Hosack et al.,“Identifying Biological Themes Within Lists of Genes with EASE,” GenomeBiol 4:R70 (2003), which is hereby incorporated by reference in itsentirety) to examine the Gene Ontology (GO) biological processannotation of WMPC-enriched genes, it was found that genes belonging tothe neurogenesis, cell adhesion, and cell communication categories wereover-represented in the WMPC profile (p<0.05, EASE score/adjusted Fisherexact test with post-hoc comparisons; Table 5).

TABLE 5 EASE Over-Represented Gene Analysis of Significantly EnrichedGenes in A2B5+ WMPC Popula- List tion EASE Bootstrap all Gene ontologycategory Hits Hits score probabilities cell adhesion 33 441 0.000 0.001cell-cell adhesion 14 133 0.000 0.001 neurogenesis 22 307 0.000 0.001morphogenesis 37 731 0.000 0.003 cell communication 83 2255 0.000 0.005organogenesis 35 687 0.000 0.005 cellular process 138 4485 0.000 0.018homophilic cell adhesion 8 56 0.000 0.021 cell migration 7 43 0.0010.032 synaptic transmission 13 199 0.005 0.178 development 47 1258 0.0050.198 transmission of nerve 13 204 0.006 0.216 impulse potassium iontransport 8 89 0.007 0.282 metal ion transport 12 202 0.013 0.451monovalent inorganic 11 183 0.018 0.539 cation transport sterolbiosynthesis 4 24 0.022 0.624 lipid metabolism 17 366 0.023 0.638 iontransport 17 370 0.025 0.675 alcohol metabolism 10 170 0.028 0.726sterol metabolism 5 48 0.033 0.786 transport 41 1194 0.035 0.818circadian rhythm 3 12 0.036 0.822 cation transport 13 268 0.039 0.838cell motility 13 269 0.040 0.847 glutamate signaling pathway 3 13 0.0410.857 secretory pathway 7 101 0.043 0.871 cell growth 6 76 0.043 0.871posttranslational membrane 3 15 0.054 0.926 targeting rhythmic behavior3 15 0.054 0.926 central nervous system 6 83 0.059 0.948 developmentcell-cell signaling 18 449 0.062 0.955 organelle organization and 14 3210.063 0.958 biogenesis microtubule-based process 7 112 0.064 0.960heterophilic cell adhesion 5 61 0.068 0.968 cytoskeleton organization 11233 0.071 0.971 and biogenesis muscle attachment 2 3 0.074 0.974 naturalkiller cell mediated 2 3 0.074 0.974 cytolysis cholesterol biosynthesis3 18 0.075 0.975 cholesterol metabolism 4 42 0.089 0.988 germ-cellmigration 2 4 0.097 0.994 glutamate transport 2 4 0.097 0.994 steroidmetabolism 6 97 0.099 0.995 Total annotated with GO 204 8027 biologicalprocess The list of significantly enriched probe sets in WMPCs wastransferred to the EASE software algorithm (version 2). Over-representedgene ontology biological process categories were determined bycomparison against the population of all Affymetrix probe sets on theU95Av2 array. Both EASE exact fisher scores and bootstrap probabilities,using 1000 iterations, were calculated. Significance cut-offs areillustrated as solid bars, at p < 0.05 bootstrap all possibilities(upper bar) and p < 0.05 EASE score (lower bar). Interestingly, celladhesion, neurogenesis and cell communication categories weresignificantly over-represented in the WMPC-specific genes.It was also noted that genes involved in sterol and cholesterolbiosynthesis were differentially expressed by the WMPC pool. Incontrast, when the same analysis was performed on genes depleted fromthe WMPC pool, it was found that genes involved in immune andinflammatory responsiveness were selectively under-represented in sortedWMPCs (Table 6).

TABLE 6 EASE Over-Represented Gene Analysis of Significantly DepletedGenes in A2B5+ WMPC Popula- List tion EASE Bootstrap all Gene ontologycategory Hits Hits score probabilities response to biotic stimulus 17685 0.000 0.001 defense response 16 631 0.000 0.001 response to externalstimulus 18 971 0.000 0.001 immune response 13 573 0.000 0.002inflammatory response 7 138 0.000 0.003 innate immune response 7 1430.000 0.003 response to chemical substance 6 143 0.001 0.022 response topest/pathogen/ 9 384 0.001 0.022 parasite response to wounding 7 2200.001 0.024 chemotaxis 5 92 0.002 0.025 taxis 5 92 0.002 0.025 antigenprocessing, exogenous 3 12 0.002 0.029 antigen via MHC class II antigenpresentation, exogenous 3 13 0.002 0.036 antigen signal transduction 201773 0.003 0.038 response to stress 11 651 0.003 0.046 antigenpresentation 3 21 0.006 0.094 antigen processing 3 21 0.006 0.094cytosolic calcium ion 3 31 0.013 0.210 concentration elevation cellcommunication 21 2255 0.018 0.256 response to abiotic stimulus 6 3490.048 0.589 G-protein signaling, coupled to 3 64 0.052 0.617 IP3 secondmessenger (phospholipase C activating) humoral immune response 4 1480.052 0.620 phosphatidylinositol-4\,5-bis- 2 11 0.061 0.685 phosphatehydrolysis humoral defense mechanism 3 87 0.088 0.831 (sensuInvertebrata) antimicrobial humoral response 3 87 0.088 0.831antimicrobial humoral response 3 87 0.088 0.831 (sensu Invertebrate)circulation 3 89 0.092 0.843 Total annotated with GO 47 8027 biologicalprocess The list of significantly depleted probe sets in WMPCs wastransferred to the EASE software algorithm (version 2). Over-representedgene ontology biological process categories were determined bycomparison against the population of all Affymetrix probe sets on theU95Av2 array. Both EASE exact fisher scores and bootstrap probabilities,using 1000 iterations, were calculated. Significance cut-offs areillustrated as solid bars, at p < 0.05 bootstrap all possibilities(upper bar) and p < 0.05 EASE score (lower bar). Several immune andinflammatory-related biological process categories were found to bedepleted from WMPC-expressed genes.

Example 11 WMPCs Express a Cohort Receptor Suggesting ActiveEnvironmental Interrogation

Belying their apparent relative quiescence, adult WMPCs were found toexpress a set of receptors that would permit their responsiveness to awide variety of both protein growth factors and neurotransmitters.Several G protein coupled receptors were differentially expressed byadult human WMPCs, the most prominent of which was the cannabinoidreceptor (CNR1) (Molina-Holgado et al., “Cannabinoids PromoteOligodendrocyte Progenitor Survival: Involvement of CannabinoidReceptors and Phosphatidylinositol-3 kinase/Akt Signaling,” J Neurosci22: 9742-9753. (2002), which is hereby incorporated by reference in itsentirety), which was confirmed to be >10-fold higher in the sorted thanunsorted cells by qPCR (p<0.01). In addition, the relativelyuncharacterized GPR19 (O'Dowd et al., “A Novel Gene Codes for a PutativeG Protein-Coupled Receptor With an Abundant Expression in Brain,” FEBSLett 394: 325-329 (1996), which is hereby incorporated by reference intheir entirety) was one of the more significantly differentiallyexpressed transcripts in these cells.

Several tyrosine kinases and phosphatases were also differentiallyexpressed (Tables 7 and 8).

TABLE 7 WMPC Enriched Genes - Tyrosine Kinase Receptors Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) TyrosineKinase Receptors ErbB3 32787_at¹ 1.60 ± 0.29 FGFR1 2056_at 2.09 ± 0.40FGFR3 31805 _(—) at 10.21 ± 4.54  IGF1R 34718_at 1.60 ± 1.68 INSR(Insulin receptor) 33162_at 1.09 ± 0.18 NTRK2 (TrkB) 1355_g_at 1.46 ±0.26 PDGFRA 1731 _(—) at 11.18 ± 0.89  Probe sets in bold indicatesignificant enrichment in WMPCs over unsorted dissociated white mattercells. ¹For genes with multiple Affymetrix probe sets, the probe setwith the most significant ratio of expression is shown.

TABLE 8 WMPC Enriched Genes - RTPβ/ζ and Related Molecules Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) qPCR (n =3-4) PTPRZ1 (RTPβ/ζ) 1364_at 8.74 ± 0.30 15.62 (10.43-23.38; p < 0.01)PTN (pleiotrophin) 234_s_at¹ 4.18 ± 0.37 4.42 (3.72-5.25; p < 0.01) SDC3(syndecan-3) 32092_at 2.69 ± 0.34 7.22 (5.92-8.81; p < 0.01) CASK31854_at 2.20 ± 0.23 4.66 (4.15-5.22; p < 0.001) ¹For genes withmultiple Affymetrix probe sets, the probe set with the mostsignificantly enriched ratio of expression is shown.Among kinases, both PDGFαR and FGFR3, the nominal high-affinity receptorfor FGF4 and FGF9, were expressed 10-fold higher by sorted WMPCscompared to the surrounding white matter. A number of other tyrosinekinases, including FGFR1, ErbB3, insulin receptor (INSR), IGF-1 receptor(IGF1R), and TrkB (NTRK2), were expressed by WMPCs, though no more sothan by their surrounding white matter. Among receptor tyrosinephosphatases, RTPβ/ζ was highly expressed and differentially so, as weremost of its known ligands (see below). A relatively uncharacterizedadenyl cyclase, adenylate cyclase 8 (ADCY8), was identified as highlydifferentially expressed, being over 17 fold higher in A2B5-sortedWMPCs.

WMPCs also expressed differentially high levels of surface receptors forseveral neurotransmitters, including both ionotropic and metabotropicreceptors for GABA, glutamate and glycine (Table 1). This suggests ahigh degree of responsiveness to the local transmitter environment andsuggests greater activity-dependent responsiveness than might have beenexpected from a nominally quiescent phenotype. In general terms, thoughthe normative roles of all of these receptors in modulating adult WMPCsis unclear, their identification presents a set of clear targets forpharmacological intervention.

Example 12 WMPCs Expressed Both Receptor Tyrosine Phosphatase β/ζ andits Ligand, Pleiotrophin

Receptor tyrosine phosphatase zeta (RTPβ/ζ) was the single mostsignificantly enriched receptor-encoding gene in this analysis, andwas >15 fold enriched in WMPCs relative to unsorted cells by qPCR (Table8; p<0.01). The Affymetrix probe set and qPCR primers were specific forthe intracellular phosphatase domain of RTPβ/ζ, as opposed to itssecreted ectodomain, phosphacan. To distinguish between the short andlong receptor isoforms of RTPβ/ζ, specific qPCR primers were designedfor each. Although both receptor isoforms were significantly moreexpressed in the WMPC, the longer isoform containing theglycosaminoglycan side chains was >25 fold enriched in WMPCs (p<0.001).

Importantly, the only known soluble ligand of RTPβ/ζ, pleiotrophin (PTN)(Meng et al., “Pleiotrophin Signals Increased Tyrosine Phosphorylationof Beta Beta-Catenin Through Inactivation of the Intrinsic CatalyticActivity of the Receptor-Type Protein Tyrosine Phosphatase Beta/Zeta,”Proc Natl Acad Sci USA 97: 2603-2608 (2000), which is herebyincorporated by reference in its entirety) was also found to beexpressed significantly higher in the WMPC-enriched profile by bothmicroarray and qPCR analysis (p<0.01). Besides binding RTPβ/ζ, PTN hasalso been shown to bind the syndecan family of transmembraneheparin-sulphate proteoglycans. Interestingly then, syndecan-3 (SDC3)mRNA was also differentially expressed by sorted adult human WMPCs, ashas been reported in rat oligodendrocyte progenitors (Bansal et al.,“Regulation of FGF Receptors in the Oligodendrocyte Lineage,” Mol CellNeurosci 7: 263-275 (1996); Winkler et al., “Syndecan-3 and Perlecan AreDifferentially Expressed by Progenitors and Mature Oligodendrocytes andAccumulate in the Extracellular Matrix,” J Neurosci Res 69: 477-487(2002), which are hereby incorporated by reference in their entirety).

Example 13 Inhibition of Tyrosine Phosphatase Activity InducesOligodendrocyte Differentiation in WMPCs

Due to the high expression of the tyrosine phosphatase receptor RTPβ/ζin WMPCs, the effect of tyrosine phosphatase inhibition on thedifferentiation of WMPCs was assessed. bpV(phen), a known potentinhibitor of tyrosine phosphatase activity, was used to induceinhibition (Posner et al., “Peroxovanadium Compounds. A New Class ofPotent Phosphotyrosine Phosphatase Inhibitors Which Are InsulinMimetics,” J Biol Chem 269: 4596-4604 (1994); Bevan et al., “SelectiveActivation of the Rat Hepatic Endosomal Insulin Receptor Kinase. Rolefor the Endosome in Insulin Signaling,” J Biol Chem 270: 10784-10791(1995); Faure et al., “Arrest at The G2/M Transition of the Cell Cycleby Protein-Tyrosine Phosphatase Inhibition: Studies on a Neuronal and aGlial Cell Line,” J Cell Biochem 59: 389-401 (1995), which are herebyincorporated by reference in their entirety). Cultures maintained for 7days exhibited a significant decline in progenitor A2B5+ cells (15±2.2%to 4±0.5%) with the addition of 25 ng/ml of bpV(phen)(n=4 patients)(FIGS. 1A-B and E). Conversely, the percentage of O4+ cells increaseddramatically (20±8.4% to 54±17.6%) when treated 25 ng/ml of bpV(phen)(n=4 patients) (FIG. 1C-E). Statistical significance was first detectedat 1 ng/ml bpV(phen) in the A2B5 positive population and 10 ng/mlbpV(phen) in the O4 positive population. The O4/A2B5 ratio rosedrastically from under 10 percent to 70 percent in response to 25 ng/mltreatment with bpV(phen) (FIG. 1E). Total cell number remained unchangedbetween dosage levels as did the number of A2B5+ BrdU+ cells, suggestingthe observed effect was due to induction of oligodendrocytedifferentiation.

Example 14 WMPCs Express Surface Adhesion Molecules that May Interactwith RTPβ/ζ

The coincident differential expression by WMPCs of both pleiotrophin andits two known receptors, RTPβ/ζ and syndecan, and the importance of bothRTPβ/ζ and syndecan-dependent signaling in transcriptional modulation,suggested the wisdom of further investigating both RTPβ/ζ and syndecanbinding partners in these cells. To this end, it was first examinedwhether WMPCs were enriched in syndecan-3 binding partners that mightsuggest its importance beyond that of a PTN sequestration moiety.Previous studies have shown that syndecan is subject to regulatedintramembrane proteolysis, that leads to the release of thePDZ-containing cytosolic protein CASK from syndecan's cytoplasmic domain(Schulz et al., “Syndecan 3 Intramembrane Proteolysis isPresenilin/Gamma-Secretase-Dependent and Modulates Cytosolic Signaling,”J Biol Chem. (2003), which is hereby incorporated by reference in itsentirety). Importantly, CASK acts as a transcriptional regulator whennot bound to syndecan; once released by syndecan, it translocates to thenucleus, where it binds to and activates the T-box family transcriptionfactor, TBR1, inducing transcription of T-box target genes (Hsueh etal., “Nuclear Translocation and Transcription Regulation by theMembrane-Associated Guanylate Kinase CASK/LIN-2,” Nature 404: 298-302(2000), which is hereby incorporated by reference in its entirety). Itwas found that CASK was indeed significantly enriched in WMPCs in boththe microarray and qPCR analyses (Table 8), suggesting the competence ofthis regulatory pathway in adult WMPCs.

It was next examined if WMPCs were enriched in RTPβ/ζ's bindingpartners. Although pleiotrophin is the only known soluble ligand forRTPβ/ζ, among other RTPβ/ζ binding partners, the extracellular matrixglycoprotein tenascin-R (TNR) (Miley et al., “High Affinity Binding andOverlapping Localization of Neurocan and Phosphacan/Protein-TyrosinePhosphatase-Zeta/Beta With Tenascin-R, Amphoterin, and TheHeparin-Binding Growth-Associated Molecule,” J Biol Chem 273: 6998-7005(1998), which is hereby incorporated by reference in its entirety), andCAM family members NrCAM (Sakurai et al., “Induction of NeuriteOutgrowth Through Contactin and Nr-CAM by Extracellular Regions of GlialReceptor Tyrosine Phosphatase Beta.” J Cell Biol 136: 907-918 (1997),which is hereby incorporated by reference in its entirety) and NCAM1(Miley et al., “Interactions of the Chondroitin Sulfate ProteoglycanPhosphacan, the Extracellular Domain of a Receptor-Type Protein TyrosinePhosphatase, With Neurons, Glia, and Neural Cell Adhesion Molecules,” JCell Biol 127: 1703-1715 (1994), which is hereby incorporated byreference in its entirety) were also differentially expressed byisolated WMPCs (Table 9).

TABLE 9 WMPC Enriched Genes - CAMs and ECM Molecules Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) CadherinsCDH11 (OB-cadherin) 36976_at 2.14 ± 0.31 CDH13 (T-cadherin) 482_at 2.40± 0.40 CDH18 (EY-cadherin) 173_at 3.03 ± 0.63 PCDH8 (Arcadlin) 32368_at4.79 ± 1.10 KIAA1775 (MT-protocadherin) 37857_at 1.96 ± 0.17 Ig-CAMsNCAM1 41289_at 2.51 ± 0.16 DSCAM 36699_at 6.94 ± 0.71 OBCAM 41093_at13.87 ± 3.28  CHL1 34193_at 11.80 ± 3.96  NRCAM 37286_at¹ 13.62 ± 2.77 Chondroitin Sulphate Proteoglycans CSPG2 (versican) 38111_at 8.36 ± 0.69CSPG3 (brevican) 32642_at 5.66 ± 0.43 CSPG4 (NG2) 38004_at 19.41 ± 2.62 CSPG5 (neuroglycan C) 39966_at 6.88 ± 1.18 Other ECM molecules TNR(Tenascin-R) 41016_at 14.66 ± 0.75  ¹For genes with multiple Affymetrixprobe sets, the probe set with the most significant ratio of expressionis shown.Indeed, virtually every described heterophilic ligand of RTPβ/ζ wasrepresented, highlighting the likely importance of in cis recognition ofRTPβ/ζ and RTPβ/ζ-dependent signaling to the maintenance of WMPCs. SinceRTPβ/ζ is able to mediate the dephosphorylation of β-catenin, whichpermits catenin translocation to the nucleus and consequentcatenin-dependent transcriptional activation (Meng et al., “PleiotrophinSignals Increased Tyrosine Phosphorylation of Beta Beta-Catenin ThroughInactivation of the Intrinsic Catalytic Activity of the Receptor-TypeProtein Tyrosine Phosphatase Beta/Zeta,” Proc Natl Acad Sci USA 97:2603-2608 (2000), which is hereby incorporated by reference in itsentirety), it would seem likely that the functions of RTPβ/ζ's bindingpartners may be to regulate RTPβ/ζ-dependent modulation of β-catenin'sbasal phosphorylation state in these cells.

Example 15 Cell-Cell Adhesion and Extracellular Matrix Molecules ofAdult Human WMPCs

Over 20 known and putative cell adhesion molecules were enriched in theWMPC mRNA pool. These included members of the cadherin, CAM, chondroitinsulfate proteoglycan (CSPG), and tenascin gene families (Table 9). Threeclassical cadherins and two protocadherins were significantly enrichedin the WMPC pool. Two type II cadherins, cadherin (CDH) 11 and 18, thatmediate homotypic Ca-dependent cell adhesion, had previously been shownto be expressed in the brain, but their cell-type specificity had beenunclear (Kimura et al., “Expression of Cadherin-11 DelineatesBoundaries, Neuromeres, and Nuclei in the Developing Mouse Brain,” DevDyn 206: 455-462 (1996), which is hereby incorporated by reference inits entirety). CDH11 can be induced by WNT activation of β-catenin,while CDH18 was initially identified as a β-catenin interacting protein(Shibata et al., “Identification of Human Cadherin-14, a Novel NeurallySpecific Type II Cadherin, by Protein Interaction Cloning,” J Biol Chem272: 5236-5240 (1997); Hadeball et al., “Xenopus Cadherin-11(Xcadherin-11) Expression Requires the Wg/Wnt Signal,” Mech Dev 72:101-113 (1998), which are hereby incorporated by reference in theirentirety). In addition, two protocadherins, PCDH8 (Arcadlin) andKIAA1775 (MT-protocadherin) (Strehl et al., “Characterization of TwoNovel Protocadherins (PCDH8 and PCDH9) Localized on Human Chromosome 13and Mouse Chromosome 14,” Genomics 53: 81-89 (1998); Nakajima et al.,“Identification of Three Novel Non-Classical Cadherin Genes ThroughComprehensive Analysis of Large cDNAs,” Brain Res Mol Brain Res 94:85-95 (2001), which are hereby incorporated by reference in theirentirety), were also selectively enriched in WMPCs. Interestingly, WMPCsalso differentially expressed the GPI-linked cadherin, CDH13, which isdown-regulated in many tumor cells and acts as a negative regulator ofEGF-stimulated neuroblastoma proliferation (Takeuchi et al., “Expressionof T-Cadherin (CDH13, H-Cadherin) in Human Brain and Its Characteristicsas a Negative Growth Regulator of Epidermal Growth Factor inNeuroblastoma Cells,” J Neurochem 74: 1489-1497 (2000), which is herebyincorporated by reference in its entirety).

The neural cell adhesion molecule, NCAM1 mRNA, was significantlyenriched in WMPCs, 2.5 fold (Table 9), in accord with the expression ofits embryonic form by rat oligodendrocyte progenitors (Grinspan et al.,“Platelet-Derived Growth Factor is a Survival Factor For PSA-NCAM+Oligodendrocyte Pre-Progenitor Cells,” J Neurosci Res 41: 540-551(1995); Ben-Hur, et al., “Growth and Fate of PSA-NCAM+ Precursors of thePostnatal Brain,” J Neurosci 18: 5777-5788 (1998), which are herebyincorporated by reference in their entirety). Several other CAM familymembers were also differentially expressed by WMPCs. These includedDSCAM, OBCAM, CHL1 and NrCAM. DSCAM (Down syndrome CAM) bindshomophilically and has been shown to be expressed in the corpus callosum(Yamakawa et al., “DSCAM: A Novel Member of the ImmunoglobulinSuperfamily Maps in a Down Syndrome Region and is Involved in theDevelopment of the Nervous System,” Hum Mol Genet 7: 227-237 (1998);Agarwala et al., “Down Syndrome Cell Adhesion Molecule DSCAM MediatesHomophilic Intercellular Adhesion,” Brain Res Mol Brain Res 79: 118-126(2000); Schmucker et al., “Drosophila Dscam is An Axon Guidance ReceptorExhibiting Extraordinary Molecular Diversity,” Cell 101: 671-684 (2000),which are hereby incorporated by reference in its entirety). OBCAM(opioid-binding CAM), has been shown to be differentially expressed byyoung oligodendroglia during early myelination (Hachisuka, et al.,“Localization of Opioid-Binding Cell Adhesion Molecule (OBCAM) in AdultRat Brain,” Brain Res 842: 482-486 (1999); Hachisuka et al.,“Developmental Expression of Opioid-Binding Cell Adhesion Molecule(OBCAM) in Rat Brain,” Brain Res Dev Brain Res 122: 183-191 (2000),which are hereby incorporated by reference in their entirety). TheL1-family member CHL1, has been shown to be expressed by A2B5⁺ ratoligodendrocyte progenitors in vitro (Hillenbrand et al., “The CloseHomologue of the Neural Adhesion Molecule L1 (CHL1): Patterns ofExpression and Promotion of Neurite Outgrowth by HeterophilicInteractions,” Eur J Neurosci 11: 813-826 (1999), which is herebyincorporated by reference in their entirety). Although its function isunclear, previous studies have highlighted the role of L1-dependentcalcium signaling in modulating the migration and survival of earlyneural progenitor cells. As noted, NrCAM may be of special interest heresince it has been shown to act as a heterophilic ligand for the RTPβ/ζectodomain (Sakurai et al., “Induction of Neurite Outgrowth ThroughContactin and Nr-CAM by Extracellular Regions of Glial Receptor TyrosinePhosphatase Beta.” J Cell Biol 136: 907-918 (1997), which is herebyincorporated by reference in its entirety).

Importantly, the extracellular matrix molecule tenascin-R (TNR) was thesecond most significantly enriched gene in the A2B5-sorted WMPC pool(Table 9). Tenascin-R has been shown to be expressed by rodent A2B5+oligodendrocyte progenitor in vitro (Jung et al., “Astrocytes andNeurons Regulate the Expression of the Neural Recognition MoleculeJanusin by Cultured Oligodendrocytes,” Glia 9: 163-175 (1993), which ishereby incorporated by reference in its entirety) and may regulate theirlineage progression (Pesheva et al., “Tenascin-R is An IntrinsicAutocrine Factor For Oligodendrocyte Differentiation and Promotes CellAdhesion by a Sulfatide-Mediated Mechanism,” J Neurosci 17: 4642-4651(1997), which is hereby incorporated by reference in its entirety). LikeNrCAM, tenascin-R also binds to the RTPβ/ζ ectodomain (Miley et al.,“High Affinity Binding and Overlapping Localization of Neurocan andPhosphacan/Protein-Tyrosine Phosphatase-Zeta/Beta With Tenascin-R,Amphoterin, and the Heparin-Binding Growth-Associated Molecule,” J BiolChem 273: 6998-7005 (1998), which is hereby incorporated by reference inits entirety), and is necessary for the normal distribution of RTPβ/ζ inwhite matter (Weber et al., “Mice Deficient for Tenascin-R DisplayAlterations of the Extracellular Matrix and Decreased Axonal ConductionVelocities in the CNS,” J Neurosci 19: 4245-4262 (1999), which is herebyincorporated by reference in its entirety). Besides thewell-characterized RTPβ/ζ binding molecules, □ four chondroitin-sulfateproteoglycans (CSPG) were differentially expressed by human WMPCs. Theseincluded versican (CSPG2), neurocan (CSPG3), NG2 (CSPG4), andneuroglycan C (CSPG5); each was enriched by 5-20 fold in A2B5-sortedWMPCs (Table 9). In addition to NG2, rodent oligodendrocyte progenitorshad previously been shown to express versican (Niederost et al., “BovineCNS Myelin Contains Neurite Growth-Inhibitory Activity Associated WithChondroitin Sulfate Proteoglycans,” J Neurosci 19: 8979-8989 (1999);Asher et al., “Versican is Upregulated in CNS Injury and is a Product ofOligodendrocyte Lineage Cells,” J Neurosci 22: 2225-2236 (2002), whichare hereby incorporated by reference in their entirety) and neurocan(Chen et al, “Inhibition of Axon Growth by Oligodendrocyte PrecursorCells,” Mol Cell Neurosci 20: 125-139 (2002), which is herebyincorporated by reference in their entirety). Yet neuroglycan C, arelatively recently cloned member of the aggrecan family localized tothe brain (Yasuda et al., “Cloning and Chromosomal Mapping of the HumanGene of Neuroglycan C (NGC), a Neural Transmembrane Chondroitin SulfateProteoglycan With an EGF Module,” Neurosci Res 32: 313-322 (1998), whichis hereby incorporated by reference in its entirety), had not previouslybeen reported to be expressed by oligodendrocyte progenitors. Remarkablythen, essentially all known brain CSPGs were differentially expressed byadult WMPCs, at many-fold higher levels than the white matter from whichthey were derived.

Example 16 WMPCs Differentially Expressed Notch-Regulated Transcripts

As noted, a number of genes characteristic of oligodendrocyteprogenitors were found differentially enriched in the A2B5+ progenitorpool. In addition though, several transcripts previously associated withless committed and early neural phenotypes were also differentiallyexpressed by these cells. Two transcription factors though restricted toneural progenitors and stem cells respectively, MASH1 (ASCL1) and HES1,were highly enriched in the WMPC pool (Table 3). MASH1 expression was12-fold greater by microarray, and >18-fold by qPCR, in A2B5⁺ cellsrelative to the unsorted white matter from which they were extracted(Table 2). HES1 was 5 fold higher by microarray, and >12 fold higher byqPCR. Both MASH1 and HES1 are downstream components of a notch signalingpathway that has already been shown to regulate oligodendrocyteprogenitor differentiation in the rat optic nerve (Wang et al., “NotchReceptor Activation Inhibits Oligodendrocyte Differentiation,” Neuron21: 63-75 (1998), which is hereby incorporated by reference in itsentirety).

A number of other notch-signaling components were expressed in WMPCs. Asthe Affymetrix U95Av2 chip does not contain probe sets to NOTCH1, it wasdetermined whether WMPCs expressed notch receptor by qPCR. NOTCH1 wassignificantly enriched in WMPCs, expressed 60% higher in WMPCs than theunsorted white matter dissociate (p<0.05, Table 10).

TABLE 10 WMPC Enriched Genes - Notch Signaling Pathway Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) qPCR (n =3-4) Notch signaling JAG1 (Jagged 1) 35414_s_at 1.26 ± 0.20 2.76(2.53-3.02; p < 0.01) JAG2 (Jagged 2) 32137_at 0.91 ± 0.13 NOTCH1 noprobe sets available 1.59 (1.50-1.70; p < 0.05) NOTCH4 39048_at 0.73 ±0.32 MSI1 (musashi 1)¹ not detected in adult samples 10.09 (7.31-13.93;n = 2, ns) NUMB 37693_at 0.59 ± 0.21 RBPSUH (RBP-J) no probe setsavailable 1.00 (0.78-1.29; n = 3, ns) FHL1 32542 _(—) at 4.50 ± 6.6510.81 (6.62-17.64; p < 0.05) FHL1B (RBP-J 9.19 (6.51-12.99; p < 0.01)binding) HES1 37393_at 5.13 ± 1.27 12.52 (11.86-13.21; p < 0.001) MASH140544 _(—) g _(—) at 12.32 ± 1.72 18.67 (15.97-21.82; p < 0.05) Genes inbold indicate significant enrichment in WMPCs over unsorted dissociatedwhite matter cells. ¹MSI1 was not detected in two of the unsortedsamples preventing calculation of an appropriate ratio and thereforereducing the sample number.NOTCH2/3 were not detected in either the WMPC or the unsorteddissociate; NOTCH4, though present, was not enriched in the WMPCs.Although notch ligands were poorly represented on the microarray,jagged1 (JAG1) was detected in both WMPCs and the unsorted dissociate(Table 10). Surprisingly, qPCR analysis revealed that WMPCs expresssignificantly more JAG1 than their surrounding white matter environment(p<0.01), suggesting the capacity for lateral inhibition ofdifferentiation among contiguous WMPCs.

Notch signaling typically activates transcription through CBF/RBP-J,which in turn up-regulates HES1 expression. In this regard, it was notedthat FHL1, a novel RBP-J binding protein, was also significantlyenriched in sorted WMPCs. FHL1 is a novel four-and-a-half LIM domaincontaining protein whose splice variant FHL1B contains an RBP-J bindingdomain (Lee et al., “Characterization of a Brain-Specific Nuclear LIMDomain Protein (FHL1B) Which is an Alternatively Spliced Variant ofFHL1,” Gene 237: 253-263 (1999), which is hereby incorporated byreference in its entirety). In the microarrays, significant expressionof FHL1 was found and by qPCR it was determined that the FHL1B splicevariant was enriched >10-fold (p<0.05).

The expression of numb protein inhibits notch signaling. The RNA-bindingprotein, musashi, binds the 3′ UTR of numb mRNA, resulting in thedown-regulation of numb protein, thereby relieving numb mediatedinhibition of notch (Imai et al., “The Neural RNA-Binding ProteinMusashi1 Translationally Regulates Mammalian Numb Gene Expression byInteracting With its mRNA,” Mol Cell Biol 21: 3888-3900 (2001), which ishereby incorporated by reference in its entirety). In WMPCs, althoughonly low levels of NUMB mRNA were found, the level of musashi1 mRNA wasmuch greater in the sorted WMPCs than in unsorted white matter cells(Table 10). Together, the differential expression of so many positiveregulators of the notch signaling pathway suggests the tonic activationof this pathway in the progenitor cell pool of the adult human whitematter.

Example 17 Components of Both Retinoid and BMP Signaling Pathways areExpressed by WMPCs

Apart from notch signaling, evidence for activation of retinoic acidsignaling and response in WMPCs was found. Retinaldehyde dehydrogenase 3(ALDH1A3), an enzyme responsible for the synthesis of retinoic acid inthe lateral ganglionic eminence (Li et al., “A Retinoic AcidSynthesizing Enzyme in Ventral Retina and Telencephalon of the EmbryonicMouse,” Mech Dev 95: 283-289 (2000), which is hereby incorporated byreference in its entirety), was enriched in WMPCs (>2 fold). This wasaccompanied by the increased expression, by >6-fold, of a syntheticretinoid-induced gene, RARRES2 (Nagpal et al., “Tazarotene-Induced Gene2 (TIG2), a Novel Retinoid-Responsive Gene in Skin,” J Invest Dermatol109: 91-95 (1997), which is hereby incorporated by reference in itsentirety), suggesting the presence of active RA signaling within theWMPC pool (Table 11).

TABLE 11 WMPC Enriched Genes - Retinoid and BMP Pathways Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) Retinoicacid signaling ALDH1A3 (RALDH3) 36686_at 2.27 ± 0.37 RARRES2 34407_at6.20 ± 1.08 BMP signaling BMP2 1113_at¹ 2.95 ± 0.25 BMP7 38515_at 4.93 ±1.18 NMA (BAMBI) 37678_at 2.55 ± 0.39 NRLN1 37630_at 14.46 ± 5.63  ¹Forgenes with multiple Affymetrix probe sets, the probe set with the mostsignificant ratio of expression is shown.

Both BMP-2 and -7 were significantly enriched in WMPCs, between 3-6 and5 fold respectively (Table 11). Along with overexpression of specificBMP ligands, expression of NMA/BAMBI (BMP and activin membrane-boundinhibitor), a negative regulator of BMP signaling whose expression isinduced in cells exposed to BMPs (Onichtchouk et al., “Silencing ofTGF-Beta Signalling by the Pseudoreceptor BAMBI,” Nature 401: 480-485(1999); Grotewold et al., “Bambi is Coexpressed With Bmp-4 During MouseEmbryogenesis,” Mech Dev 100: 327-330 (2001), which are herebyincorporated by reference in their entirety), was found. In addition,neuralin/ventropin (NRLN1), a selective antagonist of BMP4 (Sakuta etal., “Ventroptin: a BMP-4 Antagonist Expressed in a Double-GradientPattern in the Retina,” Science 293: 111-115 (2001), which is herebyincorporated by reference in its entirety), was also noted to be highlyexpressed. This observation was confirmed by qPCR, by which NRLN1was >20-fold higher in WMPCs (p<0.05). This pattern of expressionsuggests an autocrine support of WMPC maintenance by BMP2 and7-dependent pathways, with a concurrent inhibition of alternative BMPs,and BMP4 in particular, by neuralin.

Example 18 FGFR3 and PDGFαR Tyrosine Kinases are DifferentiallyExpressed by WMPCs

FGF signals have long been known to influence proliferation anddifferentiation of oligodendrocyte progenitors (for review see (Bansalet al., “Regulation of Oligodendrocyte Differentiation by FibroblastGrowth Factors,” Adv Exp Med Biol 429: 69-77 (1997), which is herebyincorporated by reference in its entirety). In adult WMPCs, it was foundthat FGFR3, though neither FGFR1 nor R2, was significantly enriched(Table 7). Indeed, the type 3 FGF receptor has previously been found tobe expressed on O4+ rodent oligodendrocyte progenitors in vitro (Bansalet al., “Regulation of FGF Receptors in the Oligodendrocyte Lineage,”Mol Cell Neurosci 7: 263-275 (1996), which is hereby incorporated byreference in its entirety). This may have significance regarding ligandcontrol of oligodendroglial mitogenesis, since it would predict thatFGFR3's cognate ligands, FGFs 1, 4 and 9, might be especiallyefficacious at directing FGFR-dependent oligodendrocytic induction andexpansion.

In this respect, it was also noted that the expression of sprouty 2(SPRY2), an inhibitor of FGFR2 signaling (Hacohen et al., “SproutyEncodes a Novel Antagonist of FGF Signaling That Patterns ApicalBranching of the Drosophila Airways,” Cell 92: 253-263 (1998), which ishereby incorporated by reference in its entirety), was increased inWMPCs relative to the unsorted population (1.96±0.26). Previous studieshave shown that SPRY2 mRNA can be induced in vitro following FGF-2signaling and can act as both an PDGF and FGF antagonist (Sasaki et al,“Identification of a Dominant Negative Mutant of Sprouty ThatPotentiates Fibroblast Growth Factor—But Not Epidermal GrowthFactor-Induced ERK Activation,” J Biol Chem 276: 36804-36808 (2001),which is hereby incorporated by reference in its entirety). Together,these data suggest an active permissiveness to FGR3 signalingconcomitant with a lack, and perhaps tonic inhibition through SPRY2, ofFGFR2 signaling.

PDGFαR was the third most significantly enriched annotated gene in WMPCs(Table 3). PDGFαR is expressed by rodent oligodendrocyte progenitors andmediates the mitogenic effect of PDGF. In addition to full length PDGFαRtranscripts, a PDGFαR splice variant that does not contain theextracellular ligand-binding domain (Mosselman et al., “DevelopmentallyRegulated Expression of Two Novel Platelet-Derived Growth FactorAlpha-Receptor Transcripts in Human Teratocarcinoma Cells,” Cancer Res54: 220-225 (1994), which is hereby incorporated by reference in itsentirety), was enriched in WMPCs (5.16±0.98).

Example 19 Sterol Biosynthesis and Metabolism

A large number of genes involved in sterol biosynthesis and metabolismwere differentially enriched in adult WMPCs (Table 12).

TABLE 12 WMPC Enriched Genes - Cholesterol Metabolism Ratio of mRNAexpression A2B5+ WMPCs:unsorted WM Affymetrix U95Av2 (n = 3) CholesterolMetabolism BASP1 32607_at¹ 1.94 ± 0.20 APOD 3668_at 5.26 ± 0.85 INSIG135303_at 2.11 ± 0.28 HMGCR 39328_at 2.38 ± 0.29 IDI1 36985_at 2.19 ±0.18 SC4MOL 33369_at 2.90 ± 0.49 LDLR 32855_at 3.21 ± 0.33 LRP1 38775_at1.75 ± 0.19 PPARG (PPARy)² 37104_1t 0.17 ± 0.13 ¹For genes with multipleAffymetrix probe sets, the probe set with the most significant ratio ofexpression is shown. ²PPARγ was significantly down-regulated in WMPCscompared to the unsorted white matter dissociate.These genes included 3-hydroxy-3-methylglutaryl-coenyzme A reductase(HMGCR), the rate-limiting enzyme in cholesterol biosynthesis, and thelow density lipoprotein receptor (LDLR), which acts to increase theavailability of intracellular cholesterol. Significantly increasedexpression of INSIG1, which encodes an intracellular regulator ofcholesterol metabolism thought to maintain pre-adipocytes in anundifferentiated state by inhibiting SREBP (Yang et al., “Crucial Stepin Cholesterol Homeostasis: Sterols Promote Binding of SCAP to INSIG-1,a Membrane Protein That Facilitates Retention of SREBPs in ER,” Cell110: 489-500 (2002); Li et al., “Insig-1 “Brakes” Lipogenesis inAdipocytes and Inhibits Differentiation of Preadipocytes,” Proc NatlAcad Sci USA 100: 9476-9481 (2003), which are hereby incorporated byreference in their entirety), was also found. Thus, cholesterolsynthetic pathways appear primed in oligodendrocyte progenitors, beforetheir terminal differentiation. In contrast, the transcription factor,PPARy, which can induce adipocyte and oligodendrocyte celldifferentiation (Walczak et al., “PPARadigms and PPARadoxes: ExpandingRoles for PPARgamma in the Control of Lipid Metabolism,” J Lipid Res 43:177-186 (2002), which is hereby incorporated by reference in itsentirety) and is expressed by both mature adipocytes andoligodendrocytes alike (Roth et al., “PPAR Gamma Activators InduceGrowth Arrest and Process Extension in B12 Oligodendrocyte-Like Cellsand Terminal Differentiation of Cultured Oligodendrocytes,” J NeurosciRes 72: 425-435 (2003), which is hereby incorporated by reference in itsentirety), was >5-fold more abundant in the unsorted white matter thanin WMPCs (Table 12). The relative scarcity of this transcript in theWMPC pool was in accord with the undifferentiated state of these cells,and suggested that PPARy expression is a concomitant of oligodendrocyticinduction from the WMPC pool.

In this study, differences in gene expression between adult human WMPCsand the white matter environment from which they derive were identified,for the purpose of defining those environmentally-responsive signalingpathways differentially operative in these cells. By comparing theexpressed RNA profiles of adult human WMPCs to those of the parentalwhite matter tissue from which each progenitor sample has beenextracted, differentially expressed genes were identified in theprogenitor pool that appeared to complement others selectively expressedby the tissue. By this means, several hitherto unpredictedligand-receptor interactions and their in cis modifiers were identified.These data suggest: 1) the importance of the RTPβ/ζ-pleiotrophin systemin WMPC self-maintenance and mobilization; 2) the potentiallyco-regulated action of syndecan-dependent CASK release in WMPCmaintenance; and 3) the role of notch signaling, as reflected by thedifferential expression of NOTCH1, HES1, musashi, and FHL1B by sortedWMPCs, in maintaining their phenotype; 4) the role of the BMP inhibitorsneuralin and BAMBI in buffering the cellular response to ambient BMPs;and 5) the likely import of FGFR3 and PDGFαR in priming these cells fordifferentiation. In the absence of FGFR3 and PDGFαR ligands in theambient white matter, these patterns of baseline gene expression mightbe expected to largely support the self-maintenance of WMPCs, whilesuppressing their differentiation.

On the basis of these data, a genomics-based model was generated for theregulatory control of adult human WMPCs, schematized here in FIG. 2. Itsmajor elements follow.

RTPβ/ζ and its Ligands are Abundantly and Selectively Expressed byWMPCs.

Receptor tyrosine phosphatase-β/ζ was the most significantly enrichedWMPC receptor gene in this analysis. Although RTPβ/ζ is expresseddevelopmentally by radial cells and neural progenitors of the fetalventricular zone (Canoll et al., “The Expression of a NovelReceptor-Type Tyrosine Phosphatase Suggests a Role in Morphogenesis andPlasticity of the Nervous System,” Brain Res Dev Brain Res 75: 293-298(1993), which is hereby incorporated by reference in its entirety), ithas also been reported to be expressed in rat oligodendrocyteprogenitors (Canoll et al., “Three Forms of RPTP-Beta are DifferentiallyExpressed During Gliogenesis in the Developing Rat Brain and DuringGlial Cell Differentiation in Culture,” J Neurosci Res 44: 199-215(1996), which is hereby incorporated by reference in its entirety).Moreover, RTPβ/ζ knock-out mice exhibit impaired recovery fromexperimental allergic encephalitis (EAE) (Harroch et al., “A CriticalRole For the Protein Tyrosine Phosphatase Receptor Type Z in FunctionalRecovery From Demyelinating Lesions,” Nat Genet 32: 411-414 (2002),which is hereby incorporated by reference in its entirety). RTPβ/ζ actsto maintain the dephosphorylated state of β-catenin, so that RTPβ/ζdeficient WMPCs might be expected to exhibit impaired wnt signaling. Inthis regard, very high levels of the secreted WNT antagonist FRZB werealso found in the WMPC pool (Table 1). FRZB has been shown to antagonizeboth WNT1 and WNT8 signaling (Wang et al., “Frzb, a Secreted ProteinExpressed in the Spemann Organizer, Binds and Inhibits Wnt-8,” Cell 88:757-766 (1997); Leyns et al., “Frzb-1 is a Secreted Antagonist of WntSignaling Expressed in The Spemann Organizer,” Cell 88: 747-756 (1997),which are hereby incorporated by reference in their entirety). Thedeficiency of RTPβ/ζ knock-out mice in remyelination, taken togetherwith the tonic expression of both RTPβ/ζ and soluble frizzled byquiescent adult human WMPCs, may suggest a role for the RTPβ/ζ-dependentdephosphorylation of β-catenin in adult WMPCs. Taken together, thesedata suggest that RTPβ/ζ signaling is required for both maintaining andmobilizing glial progenitor cells in the adult human brain.

bpV(Phen) Inhibition of RTPβ/ζ Induced Oligodendrocyte Differentiation

bpV(phen) is a potent inhibitor of tyrosine phosphatase activity (Posneret al., “Peroxovanadium Compounds. A New Class of Potent PhosphotyrosinePhosphatase Inhibitors Which Are Insulin Mimetics,”. J Biol Chem 269:4596-4604 (1994); Faure et al., “Arrest at The G2/M Transition of theCell Cycle by Protein-Tyrosine Phosphatase Inhibition: Studies on aNeuronal and a Glial Cell Line,” J Cell Biochem 59: 389-401 (1995);Bevan et al., “Selective Activation of the Rat Hepatic Endosomal InsulinReceptor Kinase. Role for the Endosome in Insulin Signaling,” J BiolChem 270: 10784-10791 (1995), which are hereby incorporated by referencein their entirety). Although bpV(phen) inhibition has been shown toinclude a range of tyrosine phosphatase receptors (Bevan et al.,“Selective Activation of the Rat Hepatic Endosomal Insulin ReceptorKinase. Role for the Endosome in Insulin Signaling,” J Biol Chem 270:10784-10791 (1995), which is hereby incorporated by reference in itsentirety), RTPβ/ζ was by far the most significantly enrichedreceptor-encoding gene in the analysis, and no other receptor tyrosinephosphatases were identified as present in WMPC isolates. Thus, it wouldbe expected that the inhibition of RTPβ/ζ was the specific incipient tooxovanadate-induced oligodendrocyte differentiation by cultured WMPCs.

Pleiotrophin Expression May Act as an Autocrine Brake Upon RTPβ/ζActivity.

Pleiotrophin inhibits RTPβ/ζ dependent-dephosphorylation of β-cateninand, by so doing, antagonizes wnt signaling (Meng et al., “PleiotrophinSignals Increased Tyrosine Phosphorylation of Beta Beta-Catenin ThroughInactivation of the Intrinsic Catalytic Activity of the Receptor-TypeProtein Tyrosine Phosphatase Beta/Zeta,” Proc Natl Acad Sci USA 97:2603-2608 (2000), which is hereby incorporated by reference in itsentirety). Besides its strong differential expression, the microarrayanalyses also revealed a number of other in cis heterophilic ligands ofRTPβ/ζ, such as NrCAM and the CSPGs, whose expression may serve tofurther modulate the phosphatase activity of RTPβ/ζ. This pattern ofgene expression suggests that parallel pathways may operate to suppresswnt signaling in adult WMPCs. Since wnt signaling can actively driveneural progenitor expansion (Zechner et al., “Beta-Catenin SignalsRegulate Cell Growth and the Balance Between Progenitor Cell Expansionand Differentiation in the Nervous System,” Dev Biol 258: 406-418(2003), which is hereby incorporated by reference in its entirety), thereversible inactivation of this pathway may be required for themaintenance of progenitors in a quiescent though mitotically competentstate.

Syndecan and CASK-Dependent Signaling Comprise a Parallel RegulatoryPathway.

The present model accommodates the expression of syndecan-3 and itsknown binding partners, a number of which—including CASK, FGFR3, andPTN—were differentially expressed by adult WMPCs. Although syndecan-3has been shown to act as a co-receptor for both PTN and FGF2, syndecan-3can also transduce extracellular signals via ligand-induced, γ-secretasemediated proteolytic cleavage of its C-terminal C2 domain (Schulz etal., “Syndecan 3 Intramembrane Proteolysis isPresenilin/Gamma—Secretase-Dependent and Modulates Cytosolic Signaling,”J Biol Chem (2003), which is hereby incorporated by reference in itsentirety). In particular, release of the C-terminal domain frees thesyndecan-3 bound protein calcium/calmodulin-activated serine kinase(CASK) to translocate to the nucleus, where it can act as atranscriptional activator through the T-box transcription factor TBR1, abrachyury family member (Hsueh et al., “Nuclear Translocation andTranscription Regulation by the Membrane-Associated Guanylate KinaseCASK/LIN-2,” Nature 404: 298-302 (2000), which is hereby incorporated byreference in its entirety). Importantly, TBR1 was indeed present in thesorted WMPCs. Although the downstream targets of TBR1 and its familymembers are largely unknown, its induction may comprise another novelsignaling pathway regulating the fate of adult WMPCs (FIG. 2).

Constitutive Activation of Notch Pathway.

The adult human WMPC resembles the rodent oligodendrocyte progenitorwith regard to the notch signaling pathway (Wang et al., “Notch ReceptorActivation Inhibits Oligodendrocyte Differentiation,” Neuron 21: 63-75(1998), which is hereby incorporated by reference in its entirety).WMPCs express high levels of both the notch receptor, NOTCH1, and itsdownstream effectors HES1 and musashi1. Indeed, the novel LIM-domaincontaining protein FHL1B, that appears to act downstream of notch tobind and transcriptionally activate RBP-J, was substantially enriched inWMPCs. Although the precise function of FHL1B in oligoneogenesis isunknown, it is worth noting that the developmental expression pattern ofthis gene clusters with that of the oligodendrocyte lineage markersPDGFαR, olig1 and olig2 during human fetal ventricular zone development.Furthermore, FHL1 expression has been described in microarray studies onskin, neural, hematopoietic, and embryonic stem cell populationssuggesting a more widespread role of FHL1 in diverse stem and progenitorcell populations (Ramalho-Santos, et al., “Sternness: TranscriptionalProfiling of Embryonic and Adult Stem Cells,” Science 298: 597-600(2002); Tumbar et al., “Defining the Epithelial Stem Cell Niche inSkin,” Science 303: 359-363 (2004), which are hereby incorporated byreference in their entirety). Surprisingly, the notch ligand JAG1 wasalso differentially expressed by adult WMPCs. During development,oligodendrocyte progenitors do not appear to express jagged (Wang etal., “Notch Receptor Activation Inhibits OligodendrocyteDifferentiation,” Neuron 21: 63-75 (1998), which is hereby incorporatedby reference in its entirety). However, its expression by adult WMPCsmay suggest a degree of lateral activation of notch signaling, that mayserve to maintain contiguous progenitors in an undifferentiated statepending mobilization (John et al., “Multiple Sclerosis: Re-Expression ofa Developmental Pathway That Restricts Oligodendrocyte Maturation,”Nature Med 8: 1115-1121 (2002), which is hereby incorporated byreference in its entirety).

Notch signaling typically results in the up-regulation of HES1, whichitself serves as a negative regulator of differentiation, as manifestedby its repression of MASH1 and OLIG2 transcription. As a result, it wassurprising to note the co-expression of MASH1 and HES1 by adult humanWMPCs. Yet although the data suggests that MASH1 and HES1 areco-expressed by single cells, it might also be the case that the WMPCpopulation contains multiple stages of parenchymal progenitor ontogeny.

The BMPs and their Antagonists.

BMP ligands can promote the differentiation of neural progenitor cellstowards an astrocytic fate, and inhibit both neurogenesis andoligodendroglial differentiation (Gross et al., “Bone MorphogeneticProteins Promote Astroglial Lineage Commitment by MammalianSubventricular Zone Progenitor Cells,” Neuron 17: 595-606 (1996); Mabieet al., “Bone Morphogenetic Proteins Induce Astroglial Differentiationof Oligodendroglial-Astroglial Progenitor Cells,” J Neurosci 17:4112-4120 (1997), which are hereby incorporated by reference in theirentirety). It has been shown that when raised at low density and highpurity, in the absence of either autocrine or paracrine growth factors,adult human WMPCs exhibit a pronounced neurogenic capacity, and are ableto differentiate into functional neurons both in vitro and, upontransplantation, in vivo (Nunes et al., “Identification and Isolation ofMultipotential Neural Progenitor Cells From the Subcortical White Matterof the Adult Human Brain,” Nat Med 9: 439-447 (2003), which is herebyincorporated by reference in its entirety). In the present study, it hasbeen shown that WMPCs express significantly more BMP2 and BMP7 than thesurrounding white matter, while expressing both membrane-bound (BAMBI)and soluble (neuralin) inhibitors of other BMPs. Although the product ofthese combinatorial interactions remains unclear, together theseobservations suggest that tonically-expressed BMPs inhibit neurogenesisat high density in vitro, and may prevent neurogenesis from WMPCs invivo.

Tyrosine Kinase Receptors.

Adult human WMPCs, like rat oligodendrocyte progenitors, respond tobasic FGF as a mitogen, and suppress terminal differentiation (Roy etal., “Identification, Isolation, and Promoter-Defined Separation ofMitotic Oligodendrocyte Progenitor Cells From the Adult HumanSubcortical White Matter,” J Neurosci 19: 9986-95 (1999), which ishereby incorporated by reference in its entirety). The present dataindicate that WMPCs express very high levels of the type 3 FGF receptor,compared to their parental white matter dissociate. Although FGFR3 haspreviously been shown to be expressed by astrocytes (Bansal et al.,“Regulation of FGF Receptors in the Oligodendrocyte Lineage,” Mol CellNeurosci 7: 263-275 (1996), which is hereby incorporated by reference inits entirety), the high level of expression in WMPCs suggests thisreceptor may provide a important target for manipulation of WMPCproliferation in vitro and in vivo. Of the three identified endogenousligands of FGFR3, FGF1 (acidic), FGF4 and FGF9, expression of FGF1 andFGF9 was detected in the microarrays (Chellaiah et al., “FibroblastGrowth Factor Receptor (FGFR) 3. Alternative Splicing inImmunoglobulin-Like Domain III Creates a Receptor Highly Specific forAcidic FGF/FGF-1,” J Biol Chem 269: 11620-11627 (1994); Hecht et al.,“Identification of Fibroblast Growth Factor 9 (FGF9) as a High Affinity,Heparin Dependent Ligand for FGF Receptors 3 and 2 but not for FGFReceptors 1 and 4,” Growth Factors 12: 223-233 (1995); Ornitz et al.,“Receptor Specificity of the Fibroblast Growth Factor Family,” J BiolChem 271: 15292-15297 (1996); Santos-Ocampo et al., “Expression andBiological Activity of Mouse Fibroblast Growth Factor-9,” J Biol Chem271: 1726-1731 (1996), which are hereby incorporated by reference intheir entirety). While both FGF-1 & -9 have been shown to be mitogenicfor A2B5-positive glial progenitors (Engele et al., “Effects of Acidicand Basic Fibroblast Growth Factors (aFGF, bFGF) on Glial Precursor CellProliferation: Age Dependency and Brain Region Specificity,” Dev Biol152: 363-372 (1992); Naruo et al., “Novel Secretory Heparin-BindingFactors From Human Glioma Cells (Glia-Activating Factors) Involved inGlial Cell Growth. Purification and Biological Properties,” J Biol Chem268: 2857-2864 (1993), which are hereby incorporated by reference intheir entirety), only FGF-1 was significantly greater in the whitematter dissociate than in the sorted WMPCs.

The PDGFαR was also highly expressed by WMPCs. PDGF is a mitogen forrodent and human glial progenitors, and can initiate oligodendrocyticdifferentiation. Moreover, PDGF signaling has been shown to inducepleiotrophin mRNA expression in 3T3 cells (Li et al., “Pleiotrophin GeneExpression is Highly Restricted and is Regulated by Platelet-DerivedGrowth Factor,” Biochem Biophys Res Commun 184: 427-432 (1992), which ishereby incorporated by reference in its entirety). This suggests thatPDGF signaling may induce oligodendrocyte commitment via autocrine PTNsignaling on RTPβ/ζ and syndecan/CASK pathways (FIG. 2).

Overview

The differentially expressed transcripts of a highly enriched progenitorcell population isolated from the adult brain have been analyzed, andthose transcripts were assessed in the context of complementary patternsof gene expression in the white matter environment. On that basis, amodel for the pathways and interactions thereof by which glialprogenitor cells are regulated in the adult human white matter has beenestablished, and by which oligodendrocytic differentiation may bedetermined. At baseline, these interactions would appear to support theself-maintenance and turnover of WMPCs, while suppressing their directeddifferentiation. As the model of FIG. 2 illustrates, these pathwaysenjoy substantial cross-talk, which might both permit the system torespond readily to environmental change, while buffering it fromperturbation by any single molecular stimulus. As such, these pathwaysmay be targeted at a number of loci for genetic or pharmacologicalmodulation of progenitor cell turnover and fate.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1-25. (canceled)
 26. A method of increasing oligodendrocyte productionfrom glial progenitor cells in a subject, said method comprising:selecting a subject requiring increased production of oligodendrocytesand administering, to the selected subject, an antagonist of receptortyrosine phosphatase-β/ζ (RPTPZ), under conditions effective toincrease, in the selected subject, oligodendrocyte production comparedto oligodendrocyte production absent said administering.
 27. The methodof claim 26, wherein the subject is an adult human.
 28. The method ofclaim 26, wherein the subject is a fetal human.
 29. The method of claim26, wherein the antagonist is selected from the group consisting ofbpV(HOpic), bpV(phen), bpV(pic), CDC25 Phosphatase Inhibitor BN82002,DMHV, Dephostatin, 3,4-Dephostatin, Phenylarsine Oxide, Protein TyrosinePhosphatase CD45 Inhibitor, Protein Tyrosine Phosphatase Inhibitor I,Protein Tyrosine Phosphatase Inhibitor II, Protein Tyrosine PhosphataseInhibitor III, Protein Tyrosine Phosphatase Inhibitor IV, RK-682, SodiumStibogluconate, and bpV(bipy).
 30. A method of treating a subject for acondition modulated by underproduction, dysfunction, or loss ofoligodendrocytes from human white matter, said method comprising:selecting a subject with a condition modulated by underproduction,dysfunction, or loss of oligodendrocytes from human white matter andadministering, to the selected subject, an antagonist of receptortyrosine phosphatase-β/ζ (RPTPZ) under conditions effective to treat thecondition modulated by underproduction, dysfunction, or loss ofoligodendrocytes.
 31. The method of claim 30, wherein the condition isselected from the group consisting of the pediatric leukodystrophies,the lysomal storage diseases, congenital dysmyelination, cerebral palsy,inflammatory demyelination, post-infectious and post-vaccinialleukoencephalitis, radiation- or chemotherapy-induced white matterdamage, and vascular demyelination.
 32. The method of claim 30, whereinthe receptor tyrosine phosphatase antagonist is selected from the groupconsisting of bpV(HOpic), bpV(phen), bpV(pic), CDC25 PhosphataseInhibitor BN82002, DMHV, Dephostatin, 3,4-Dephostatin, PhenylarsineOxide, Protein Tyrosine Phosphatase CD45 Inhibitor, Protein TyrosinePhosphatase Inhibitor I, Protein Tyrosine Phosphatase Inhibitor II,Protein Tyrosine Phosphatase Inhibitor III, Protein Tyrosine PhosphataseInhibitor IV, RK-682, Sodium Stibogluconate, and bpV(bipy).
 33. Themethod of claim 30, wherein the subject is an adult human.
 34. Themethod of claim 30, wherein the subject is a fetal human.